THE 


TECHNOLOGY 


BACTERIA  INVESTIGATION 


EXPLICIT    DIRECTIONS    FOE    THE 
STUDY  OF  BACTERIA 


CULTURE,  STAINING,  MOUNTING,  ETC.,  ACCORDING  TO 

THE  METHODS  EMPLOYED  BY  THE  MOST 

EMINENT  INVESTIGATORS 


BY 

CHARLES   S.  DOLLEY,  M.D. 
\\ 


S.  E.   CASSINO  AND  COMPANY 

1885 


Copyright,  by 
S.  E.  CASSINO  AND  COMPANY, 


ELECTROTYPED  BY 
C.  J.  PETEBS  AND  SON,  BOSTON. 


PEBFACE. 


THE  following  pages  are  presented  with  the 
hope  that  they  will  stimulate  careful  study  of 
the  Schizomycetes  by  American  investigators,  and 

'       •  •  v 

assist  them  in  adding  their  share,  as  they  have 
done  in  other  branches  of  scientific  research, 
to  the  mass  of  facts  concerning  Bacteria  ;  which, 
with  two  or  three  exceptions,  have  been  thus  far 
brought  to  light  by  European  students. 

C.  S.  D. 

NAPLES  ZOOLOGICAL  STATION,  April  3,  1885. 


TABLE  OF  CONTENTS. 


Part  I. 

GENERAL  DIRECTIONS 1 

I.  MICROSCOPICAL  PREPARATIONS 2 

The  Study  of  Living  Forms 3 

Schnetzler's  Method  of  obtaining  Bacteria  from  the  Air         .  7 

Brautlecht's  Method  of  obtaining  Bacteria  from  the  Air         .  8 

Miquel's  Method  of  obtaining  Bacteria  from  the  Air        .        .  8 

Miquel's  Second  Method  for  the  Analysis  of  Rain    .        .        .  9 

Miquel's  Method  for  the  Analysis  of  Drinking  Water     .        .  10- 
Miquel's  Method  for  ascertaining  the  Hourly  Variations  of 

Aerial  Bacteria 11 

Hesse's  Method  for  the  Quantitative  Estimation  of  Micro- 
organisms contained  in  the  Air         ......  15 

Marchand's  Method  for  the  Examination  of  Microbes  or  Cor- 
puscles held  in  Suspension  in  Water 16 

Literature  of  Bacteria  contained  in  Air,  Water,  or  Earth       .  17 
Naegeli's  Method  for  the  Isolation  of  Particular  Bacterial 

Forms 22 

Buchner's  and  Robert's  Method  for  obtaining  Bacillus  Subtilis  23 
Tiegel's   Method   for  proving  the  Existence  of  Bacteria  in 

the  Tissues  of  Healthy,  Living  Organisms    ....  23 
Burdon-Sanderson's   Method   for  proving  the  Existence  of 

Bacteria  in  the  Tissues  of  Healthy  Living  Organisms    .        .  24 
Chiene  and  E wart's  Method  for  proving  the  Existence  of 

Bacteria  in  the  Tissues  of  Healthy  Living  Organisms    .        .  25 
Nencki  and  Giacosi's  Methods  for  proving  the  Existence  of 

Bacteria  in  Healthy  Living  Organisms 25 

Literature  on  Bacteria  in  Healthy  Living  Organisms       .        .  27 

To  obtain  Blood  for  Microscopical  Examination        ...  32 

Moss's  Method  for  obtaining  Blood  for  Investigation       .        .  32 

To  obtain  Fluid  or  Serum  from  a  freshly  extracted  Organ      .  33 

V 


VI  TABLE   OF   CONTENTS. 

The  Study  of  Microscopical  Preparations  of  Fixed  and  Hard- 
ened Bacteria 34 

The  Use  of  Staining  Fluids 35 

Hazel  wood's  General  Blue  Stain 37 

Blanchard's  Method  of  Staining  "  Proliferous  Membranes,"  37 

"T.  C.'s"  Method 38 

•  Cover-glass  Preparations 39 

General  Method  for  Staining  Cover-glass  Preparations    .        .  41 

Koch's  Method  for  Staining  Cover-glass  Preparations      .        .  41 

Gram's  Method  for  Staining  Cover-glass  Preparations     .        .  42 

The  Preparation  of  Bacteria  in  and  upon  Firm  Substances     .  43 

Weigert's  Method 44 

Schutz's  Method 45 

Babe's  Method 45 

Subbotin's  Combination  of  Staining  Fluids       ....  46 

Mounting 46 

Preparation  of  Bacteria  for  Photographing        ....  48 

Sternberg's  Method 48 

Kaschka's  Method 48 

Dufrenne's  Method 49 

Literature  of  the  Preparation  and  Investigation  of  Bacteria 

in  General 51 

II.  CULTURE  EXPERIMENTS 56 

Fluid  Culture  Media 59 

Pasteur's  Sterilization  Method 60 

Buclmer's  Sterilization  Method 61 

Tyndall's  Sterilization  Method  by  Discontinuous  Heating  .  61 

MiqueFs  Sterilization  Method  without  Heat  ....  61 

Gautier's  Method  of  Sterilization  without  Heat  ...  62 

Koch,  Gaffky,  and  Loeffler's  Steam  Sterilizing  Cylinder  .  63 

Solid  or  Gelatine  Culture  Media 64 

Koch's  Serum  Culture  Medium 66 

Culture  Vessels 67 

Salmon's  Culture  Tube 67 

Sternberg's  Culture  Flasks 70 

To  introduce  the  Sterilized  Culture  Fluid  into  the  Sterilized 

Culture  Vessel 71 

To  sow  the  Microbes  in  the  Culture  Medium  ....  72 

To  carry  on  the  Cultures 73 

Van  Tieghem  and  Lemonnier's,  and  Miquel's  Methods  for 

the  Cultivation  of  Bacteria  upon  the  Slide    ....  73 


TABLE   OF   CONTENTS.  Vli 

Salmonsen's  Method  for  Pure  Cultures 75 

Literature  on  Culture  Methods 76 

III.  VACCINATION  OR  INOCULATION  EXPERIMENTS         .        .  78 
Literature  of  Inoculation  Methods 81 

IV.  BIOLOGICAL  ANALYSIS 82 

Eiigelmann's  Method  for  Studying  the  Effect  of  Light  on 

Certain  Bacteria 85 

Eiigelmann's  Method  for  the  Determination  of  a  Chromophyll 

Assimilating  Power  in  any  given  Bacteria  ....  86 
Duclaux's  Method  for  Studying  the  Effect  of  Sunlight  upon 

the  Germs  of  Bacteria 87 

Pictet  and  Yung's  Method  for  Ascertaining  the  Action  of  Cold 

upon  Microbes 88 

Literature  on  the  Biology  of  Bacteria 88 


Part  H. 

SPECIAL  METHODS  FOR  INVESTIGATING  PATH- 
OGENIC BACTERIA 93 

ANTHRAX 99 

Toussaint's  Method 99 

Pasteur's  Method 100 

Chaveau's  Methods 103 

Chamberland  and  Roux's  Method 106 

Koch,  Gaffky,  and  Loemer's  Method 107 

Weigert's  Staining  Method 109 

Feltz's  Method  of  Ascertaining  the  Role  of  Earthworms  in 

the  Propagation  of  Anthrax 109 

Literature 109 

CHOLERA 117 

Koch's  Methods 118 

Nicati  and  Rietsch's  Method 126 

Bochefontaine's  Method 126 

Literature 127 

GLANDERC 131 

Loeffler  and  Shutz's  Method         .......  131 

Literature       .  ,132 


Vlll  TABLE   OF  CONTENTS. 

HOG  CHOLERA 133 

Literature 134 

HYDROPHOBIA 135 

Gibier's  Method  of  Attenuation 135 

Pasteur's  Method  of  Inoculation          .        .        .        .        .        .135 

Babe's  Staining  Method 137 

Literature 139 

LEPROSY 140 

Babe's  Staining  Method 141 

Baumgarten's  Staining  Method 141 

Neisser's  Methods 142 

Literature 143 

MALARIA 145 

Klebs  and  Tommassi-Crudelli's  Method  for  Collecting  Ma- 
laria Germs 145 

Richard's  Method  for  the  Study  of  the  Blood   .        .        .        .147 

Literature 147 

MALIGNANT  OEDEMA 150 

Pasteur's  Method 150 

Literature 150 

SEPTIC^MIA  OF  DOMESTIC  MOUSE 151 

SEPTICAEMIA  OF  RABBITS 152 

Literature 153 

SYPHILIS 153 

Birsch-Hirschf eld's  Method 153 

Kleb's  Method 153 

Literature 154 

TUBERCULOSIS 155 

Koch's  Method 156 

Ehrlich's  Methods .  157 

Ziehl's  Method -     .        „        „  158 

Balmer  and  Fraenzel's  Methods 159 

Rindfleish's  Method 159 

Orth's  Method 160 

Lichtheim's  Method 161 

Petri's  Method 161 

Gibb's  Methods              163 

Baumgarten's  Methods         ........  164 


TABLE   OF  CONTENTS.  ix 

Weigert's  Method 166 

Fraenkel's  Method 166 

Pfuhl-Petri's  Method 167 

Senkewitch's  Method 168 

Raatzer's  Method 168 

Long's  Method 169 

Peter's  Method 169 

Veraguths'  Method 170 

Coze  and  Simon's  Staining  Methods 171 

Deje'rine's  for  Study  of  Concrements  from  Lungs    .        .        .  171 
Giboux's  Method  of  Testing  the  Inoculability  of  Tubercu- 
losis through  Respiration 172 

Ermengen's  Staining  Method 172 

Brun's  Staining  Method 173 

Burrill's  Staining  Method 174 

Hartzell's  Staining  Method 174 

Quinlan's  Staining  Method 175 

Negri's  Method  for  Staining  the  Spores 176 

Beinstadler's  Method 178 

Celli  and  Guarnieri's  Method  for  Ascertaining  whether  the 
Microbes  of  Tuberculosis  are  thrown  into  the  Air  by  Expira- 
tion   .179 

Literature 181 

TYPHOID  FEVER 193 

Letzerich's  Method 193 

Rindfleish's  Method  for  Contaminated  Well-water  .        .        .195 

WHOOPING  COUGH 197 

Burger's  Method 197 

Literature 197 

CONCRETIONS  OF  THE  LACHRYMAL  DUCTS        ....  197 

Literature 197 

DENTAL  CARIES 198 

Leber's  Method 198 

Miller's  Method 199 

Literature 199 

AREA  CELSII 200 

Buchner's  Method  of  Study 200 

Von  Sehlen's  Method 200 

Literature                                                                                       .  201 


X  TABLE   OF   CONTENTS. 

CHICKEN  CHOLERA 201 

Barthelmy's  Method  of  Study 202 

Pasteur's  Method 202 

Literature 203 

DIPHTHERIA 204 

Loeffler's  Method 204 

Literature 205 

ERYSIPELAS 207 

Fehleisen's  Methods .        .207 

Literature 207 

FURONCLE 208 

Pasteur's  Method 209 

Literature 209 

GONORRHCEA 210 

Neisser's  Methods 210 

Literature 211 

OSTEOMYELITIS 213 

Krause's  Methods 213 

Literature 214 

PNEUMONIA-CROUPOSA 215 

Friedlaender's  Methods 215 

Literature 217 

RECURRENT  FEVER 220 

Koch's  Method 220 

Friedlaender's  Method  of  Obtaining  the  Spirilla      .        .        .220 

Literature 221 

YELLOW  FEVER 222 

Friere's  Methods 222 

Literature 222 

MISCELLANEOUS  PATHOGENIC  BACTERIA,  WITH  LITERATURE  223 

Contagious  Septicaemia 22,  > 

Rheumatic  Arthritis 225 

Endocarditis  Ulcerosa 225 

Infectious  Wound  Diseases 226 

Variola '  .  227 

Influenza  Epidemica 229 

Meningitis 229 

Measles .230 


TABLE  OF  CONTENTS.  xi 

Phlegmon 230 

Scarlet  Fever 230 

Molluscum  Contagiosum 231 

Dilatation  and  other  Diseases  of  the  Stomach  .        .        .        .231 

Anaemia  Perniciosa 232 

Ulcerative  Stomatitis  in  the  Calf .232 

Cattle  Plague 232 

Acute  Yellow  Atropy  of  the  Liver 232 

Dysentery 233 

Ozaena 233 

Foot  Rot 233 

Verruga  Peruana 233 

Diabetes 233 

Chyluria 234 

Foetid  Feet 234 

Malleus  humidus 234 

Pyaemia 234 

Diseases  of  Insects 234 

BAQTERIA  IN  PLANT  TISSUES 235 

Ducleaux's  Method  of  Studying  the  Effect  of  Microbes  upon 

Germination 235 

Ralph's  Method  for  Demonstrating  the  Presence  of  Microbes 

in  the  Cells  of  Aquatic  Plants 235 

Literature  .  236 


Part  JHHE. 

FORMULARY 238 

Bergmann's  or  Bucholz's  Fluid 238 

Bismarck-Brown 238 

Glycerine  Aniline-Brown 239 

Vniline  Oil 239 

Aniline  Water 240 

Aniline-Yellow 240 

Acidulated  Fluids  for  Decolorizing 240 

Ether 241 

Alcohol 241 

Canada  Balsam 241 

Chrysoidin 241 


Xll  TABLE  OF  CONTENTS. 

Cement  for  Glycerine  Mounts 242 

Culture  Fluids 242 

Eosin  Haematoxylin 243 

Eosin 243 

Fuchsin 244 

Fuchsin-Aniline-Oil 245 

Gentian-Violet 245 

Kleb's  Glycerine-Jelly 246 

Glycerine 246 

Boehmer's  ILematoxylin 247 

Methyl-Blue .247 

Methyl-Green  and  Malachit-Green 248 

Methyl-Violet 248 

Magenta 249 

Nigrosin  or  Aniline-Black 249 

Orseille 250 

Oil  of  Cloves 250 

Osmic  Acid 250 

Peptone  Solution 251 

Picrocarmine          ..........  251 

Picrocarminate  of  Ammonia 252 

Acetate  of  Potash  for  Mounting 252 

Caustic  Potash  Solutions 253 

Rosaniline  Chlorohydrate 253 

Acid  Fuchsin 253 

Vesuvin 254 

Plaster  of  Paris  Filters 254 

Cleaning  Fluids 254 

Soap  Imbedding  Mass .255 

Celloidin  Imbedding  Mass 256 

Brun's  Mounting  Medium 258 

Gautier's  Red  Lead  Cement        .  * 259 

Nutritive  Paper 259 

Solid  Culture-Media 260 

Lichen  Jelly 260 

Hydrocele  Fluid 261 

Blood  Serum          .        . 262 

Agar  Agar 262 

Gelatine  ....  .263 


BACTERIA  INVESTIGATION. 


PART  I. 

GENERAL    DIRECTIONS. 

IN  the  study  of  the  Schizomycetes  it  will  be 
found  of  advantage  to  use  slides  of  the  whitest 
possible  glass,  of  from  1  to  2  mm.,  and  cover- 
glasses  of  from  0.10  to  0.18  mm.  in  thickness.  It 
is  of  the  highest  importance  that  these,  as  well 
as  all  other  implements,  such  as  tubes,  pincers, 
needles,  wires,  etc.,  should  be  made  perfectly 
clean  immediately  before  their  use.  Metallic  in- 
struments can  be  best  sterilized  by  heating  in  the 
flame  of  a  spirit  lamp  or  Bunsen  burner.  Slides 
and  cover-glasses  should  be  kept  in  a  cleaning 
fluid  [F.  40] ,  and  washed  and  wiped  well  before 
using.  Other  glassware  may  be  cleaned  by  the 
use  of  this  same  fluid,  or  by  passing  it  through 
concentrated  hydrochloric  acid  and  afterwards 
washing  it  in  hot  alcohol. 

All  stain  ing-fluids  should  be  filtered  through 
good  Swedish  filter-paper  previous  to  use,  and 


2  THE  TECHNOLOGY   OF 

if  much  importance  is  attached  to  the  investi- 
gation, the  filtrate  should  be  examined  microscopi- 
cally. 

The  presence  and  variety  of  Bacteria  may,  by 
the  trained  eye,  be  diagnosed  with  objectives  of 
medium  power  [230  :  540  diam.],  but  in  the  study 
of  pathogenic  micro-organisms  a  good  homogene- 
ous immersion  objective  and  an  Abbe's  condensor 
are  considered  essentials.  The  best  light  is  that 
obtained  from  white  clouds,  or  sunlight  reflected 
from  a  white  wall ;  next  to  this  comes  the  light 
obtained  from  good  kerosene,  which  is  improved 
by  the  addition  of  a  small  piece  of  camphor.  To 
protect  the  eyes  from  the  glare  of  the  light,  Engel- 
mann  regards  a  green  or  greenish-blue  glass  placed 
between  the  object  and  the  light  as  preferable  to 
the  cobalt-blue  glass  ordinarily  used,  or  to  a  blue 
lamp  chimney,  while  Flesch  considers  a  pale  yel- 
low glass  better  than  the  green.  In  no  case  should 
the  colored  glass  be  placed  between  the  object  and 
the  eye.  In  searching  for  Bacteria  in  any  organ, 
it  is  better  first  to  study  such  fluids,  serous  or 
other,  as  may  be  obtained  from  the  organ,  and 
then  the  organ  itself.  The  methods  used  for  the 
study  of  Bacteria  may  be  grouped  under  four 
heads,  i.e.,  I.  Microscopical  preparations;  II. 
Culture  experiments ;  III.  Vaccination  or  Inocu- 
lation experiments  ;  IV.  Biological  analysis. 


BACTERIA    INVESTIGATION.  3 

I.  MICROSCOPICAL  PREPARATIONS. 
In  this  method  the  microbes l  are  to  be  distin- 
guished from  similar  inorganic  particles,  either  by 
the  characteristic  form  of  single  individuals,  by 
the  arrangement  of  these  into  characteristic  groups 
or  colonies,  or  by  their  relation  to  chemical  re- 
agents. It  must  be  borne  in  mind  that,  with  few 
exceptions,  all  Bacteria  at  different  stages  of  their 
life  history  exhibit  different  forms.  The  spores 
or  germs  of  all  Bacteria  are  round,  micrococcus- 
like  bodies,  from  which  develop  rod-like  forms, 
spiral  or  straight  filamentous  forms,  and  eventu- 
ally the  forms  typical  of  the  species.  We  may 
study  these  in  microscopical  preparations,  either 
alive  in  some  transparent  nourishing  medium,  or 
after  killing  and  fixing  them.  The  first  of  these 
methods,  i.e.,  The  Study  of  Living  Forms,  is 
not  best  adapted  for  persons  unacquainted  with 
the  various  bacterial  types.  These  should  first  be 


1  The  word  "  microbe  "  was  introduced  by  Mr.  Chas.  Se'dillot  as 
follows  :  — 

"  Les  noms  de  ces  organismes  sont  tres  nombreux  et  devront  etre 
definis  et,  en  partie,  reforme's.  Le  mot  microbe  (Mi%p6s,  petit, 
Bi'oj,  vie)  ayant  1'avantage  d'etre  plus  court  et  d'une  signification 
plus  generate,  et  mon  illustre  ami,  M.  Littre,  le  linguiste  de  France 
le  plus  competent,  1'ayant  approuve',  nous  1'adoptons,  sans  nean- 
moins  renoncer  a  ceux  en  usage,  pour  la  designation  de  varie'tes 
plus  particulierement  etudie'es."  Vid.  "  De  1'influence  des  d^cou- 
vertes  de  M.  Pasteur  sur  les  progres  de  la  chirurgie."  Compt. 
Rend.  T.  86,  p.  634. 


4  THE  TECHNOLOGY   OF 

rendered  familiar  by  the  study  of  fixed  and  stained 
preparations.  When  this  has  been  accomplished, 
the  living  microbes  may  be  studied  for  the  pur- 
pose of  learning  their  peculiarities  of  movement, 
grouping,  behavior  towards  various  reagents,  and 
the  finer  details  of  structure  lost  by  drying,  etc., 
by  placing  upon  a  properly  cleaned  cover-glass  a 
drop  of  the  fluid  containing  them,  and  inverting  it 
over  a  shallow  cell  or  the  cavity  of  a  hollowed-out 
slide,  or  by  a  thin  layer  of  the  fluid  spread  out 
and  covered  upon  an  ordinary  slide.  If  too  large  a 
number  of  microbes  are  in  the  field  at  once,  pro- 
ducing a  confused  picture,  the  liquid  may  be 
thinned  by  the  addition  of  a  drop  of  pure  filtered 
water,  common  salt  solution,  or  peptone  solution 
[F.  31].  The  investigator  is  not  obliged  to 
search  far  for  specimens  of  Bacteria  upon  which  to 
institute  comparative  studies.  The  atmosphere  of 
the  laboratory,  of  the  home,  of  city  and  country, 
as  has  been  shown  by  Pasteur,  Miquel,  and  others, 
is  a  rarely  failing  source  of  living  germs,  if  not 
of  developed  microbes.  Flowing  and  stagnant 
water,  both  fresh  and  salt,  affords  respectively 
peculiar  forms  ;  the  so-called  "  filth  "  Bacteria  may 
be  sought  in  infusions  of  decaying  animal  or  vege- 
table matter ;  those  forms  which  act  as  special  fer- 
ments will,  of  course,  be  found  in  fluids  undergoing 
butyric,  lactic,  or  other  fermentation,  as  the  case 
may  be. 


BACTEEIA   INVESTIGATION.  5 

The  various  fluids  of  living  animal  bodies  in 
health  and  disease  afford  an  abundant  harvest  of 
microbes  illustrating  nearly  every  group  of  the 
Schizomycetes. 

Among  invertebrate  animals  Metschnikoff  found 
in  the  serous  fluids  large  numbers  of  Bacteria, 
which  were  being  eagerly  pursued  and  devoured 
by  the  amseboid  cells  of  the  animal.  In  fishes 
Richtet  has  shown  that  the  blood  and  lymph  con- 
tain Bacteria.  The  secretions  of  healthy  human 
beings  have  been  shown  by  Sternberg  and  others 
to  abound  in  a  variety  of  forms ;  thus  the  saliva 
has  repeatedly  been  demonstrated  to  be  rich  in 
micro-organisms  of  great  variety,  long  Leptothrix 
threads  of  different  thickness,  round  Micrococci  of 
varying  size,  in  heaps  and  chains ;  often  Bacilli 
and  beautiful  Spirochcele.  All  these,  together  with 
numerous  specimens  of  the  lower  orders  of  Fungi, 
may  be  examined  by  preparing  as  above,  or  after 
being  fixed  and  hardened,  as  directed  further  on. 
The  vaginal  secretions,  like  those  of  the  mouth, 
furnish  a  nidus  for  microbes  of  great  variety.  In 
the  stomach  the  ordinary  bacterial  forms  are  found 
as  a  rule,  only  in  small  numbers,  owing  probably 
to  the  acidity  of  the  digestive  juices.  The  intes- 
tine, on  the  other  hand,  is  rich  in  Bacteria,  Micro- 
cocci  and  Bacilli  of  all  sizes  and  shapes ;  among 
the  latter,  at  times,  the  ileum  and  colon  contain 
specimens  of  B.  amylobacter,  the  source  of  bu- 


6  THE   TECHNOLOGY  OF 

tyric  acid  fermentation.  Besides  the  above,  which 
appear  in  their  fully-developed  condition,  Billroth, 
Nencki,  and  others  have  proven  conclusively  that 
the  tissues  of  living,  healthy  animals  contain  my- 
riads of  bacterial  germs  or  spores,  while  patholo- 
gists,  in  their  study  of  the  diseased  body,  have 
indicated  the  presence  of  many  peculiar  forms, 
some  of  which  are  regarded  as  the  specific  agents 
of  infectious  disease.  Thus  the  lachrymal  ducts 
and  the  sweat  are  at  times  inhabited  by  two  very 
different  salt-loving  forms  ;  the  vaginal  and  urethral 
secretions,  as  well  of  those  of  the  eyes,  become 
at  times  the  home  of  the  gonorrhceal  microbe  ;  the 
stomach,  in  cases  of  catarrh,  dilatation,  and  cancer, 
is  often  occupied  by  Sarcina,  which  also,  along  with 
other  Bacteria,  makes  its  home  in  the  vermiform 
appendix  of  our  domestic  fowls.  The  intestine  of 
the  typhoid  fever  or  cholera  patient  furnishes  the 
Bacilli  peculiar  to  these  diseases.  The  lymph  in 
syphilis,  measles,  erysipelas,  etc.,  shows  other 
forms,  and  the  blood  of  diphtheria  patients  and 
those  suffering  from  recurrent  fever  still  others. 
Others  take  up  their  abode  in  various  organs,  as  has 
been  shown  in  leprosy  and  tuberculosis ;  the  pus 
of  abscesses  —  tubercular,  furuncular,  and  other- 
wise— present  Micrococci  and  Bacilli  m  abundance, 
while  decayed  teeth  are  already  filled  with  an  amal- 
gam of  Bacteria. 

The  enumeration  of  pathogenic  Bacteria  is,  how- 


BACTERIA  INVESTIGATION.  7 

ever,  at  this  point  out  of  place,  it  being  only  intend- 
ed here  to  indicate  the  success  which  surely  awaits 
the  seeker  after  microbes  in  healthy  and  diseased 
animals.  All  or  any  of  these  forms  may  be  studied 
as  microscopical  preparations,  alive,  by  the  simple 
methods  we  have  indicated,  or  they  may  be  sub- 
jected, while  under  the  cover-glass,  to  a  number 
of  manipulations,  which  will  be  fully  described 
under  "Biological  Analysis" 

BACTEKIA  OF  THE  AIR,  WATER,  AND  EARTH. 

Although  Bacteria,  as  we  have  seen,  are  almost 
omnipresent,  experience  has  shown  that  certain 
methods  are  to  be  preferred  in  bringing  them  into 
position  for  microscopic  study.  Thus  it  has  been 
found  that  in  the  study  of  Aerial  Bacteria  the  or- 
dinary methods  employed  for  collecting  the  dust  of 
the  atmosphere  are  of  slight  use,  since  it  is  difficult 
to  distinguish  the  germs  among  a  large  number  of 
inanimate  granules  and  in  a  miscellaneous  mixture 
of  debris. 

8chnetzler  points  out  that  we  have  at  our  com- 
mand, for  the  study  of  aerial  germs,  a  small  appa- 
ratus traversed  by  about  8,000  cubic  centimeters  of 
air  per  minute,  which  may  inform  us  as  to  these 
floating  germs.  This  is  no  other  than  the  nasal 
cavity,  on  the  mucous  surface  of  which  air  particles 
are  deposited.  To  observe  these  he  advises  in- 
jecting the  nose  with  distilled  water  (completely 


8  THE   TECHNOLOGY   OF 

sterilized)  by  means  of  a  glass  syringe  previously 
calcined.  The  liquid  thus  obtained  is  to  be  sub- 
jected to  examination  by  any  or  all  the  methods 
employed  for  the  microscope,  or  culture. 

BrautlecMs  Method,  employed  in  the  course  of  a 
research  on  the  presence  of  Bacteria  in  the  effluvia 
and  vapors  of  fever  districts,  was  to  mix  baked 
sand,  gritty  earth,  and  tolerably  loamy  garden 
mould,  with  liquids  containing  Bacteria,  and  then 
covering  the  mixture  with  a  bell-glass,  using  all 
ordinary  precautions  against  external  contamina- 
tion. After  a  few  hours  he  found  in  the  vapors 
condensed  under  the  bell-glass  a  large  number  of 
microbes,  of  the  same  form  invariably  as  those 
contained  in  the  liquid  used. 

MiqueTs  Method  of  obtaining  Bacteria  from  the 
air  is  by  the  use  of  rain,  which  contains  more 
microbes  than  the  water  condensed  artificially  from 
the  atmosphere  by  means  of  "  aeroscopes,"  by  ven- 
tilators or  other  appliances.  He  employs  an  appa- 
ratus which  he  calls  an  "  Udometer."  An  iron  rod 
is  fastened  horizontally  to  the  upper  portion  of  a 
wooden  post  set  in  the  ground  at  a  distance  from 
any  trees  or  habitation,  where  it  will  receive  the 
first  drops  of  rain.  Two  L-shaped  arms,  each  ter- 
minating at  one  end  in  a  ring  and  arranged  at  the 
other  to  slide  upon  the  stationary  rod  and  fasten 
with  a  thumbscrew,  like  the  ordinary  filter  stands 
of  the  chemist,  are  provided.  In  the  ring  of  one  of 


BACTEKIA  INVESTIGATION.  9 

these  is  placed,  after  being  thoroughly  sterilized  at 
a  high  temperature,  a  nickel-  or  silver-plated  fun- 
nel. In  the  second  ring,  which  is  arranged  to  hang 
directly  below  the  first,  is  placed  a  platinum  cru- 
cible, likewise  sterilized,  and  for  which  a  close-fit- 
ting cover  is  provided.  By  means  of  this  appara- 
tus, rain  may  be  collected  at  the  beginning,  in  the 
midst,  or  at  the  latter  part  of  a  storm,  although 
the  first  rain  contains  the  most  bacterial  germs. 
The  cover  being  adjusted  to  avoid  contamination 
during  transport,  the  water  is  taken  to  the  labora- 
tory, w^here  the  Bacteria  or  their  spores,  which  have 
been  washed  from  the  air,  may  be  examined  as 
microscopical  preparations  or  by  any  of  the  other 
methods  of  study. 

MiqueTs  Second  Method  for  the  analysis  of 
rain.  For  the  analysis  of  rain,  Dr.  Miquel  has 
invented  a  special  apparatus, — his  "  Udobacte- 
riemetre," — consisting,  in  brief,  of  a  glass  shade 
with  its  neck  fitted  with  a  stopper,  through  which 
is  passed  the  long  stem  of  a  metal  funnel  that  de- 
livers, by  droplets,  the  rain  caught,  and  projects 
them  upon  the  nutritive  paper  [F.  45]  arranged  in 
a  very  wide  truncated  cone,  which  is  rotated  at  some 
speed  by  clockwork  beneath.  The  droplets  are 
carried  by  the  rotation  a  little  way  along  the 
sterilized  paper  cone,  moistening  the  lichen  surface 
in  their  course  at  different  points, —  no  troublesome 
liquefaction  taking  place,  as  with  ordinary  gelatine. 


10  THE   TECHNOLOGY   OF 

The  cone  of  paper,  at  the  end  of  the  experiment, 
is  placed  in  the  incubating  stove  with  its  damp 
atmosphere,  and  affords  excellent  results. 

MiqueTs  Method  for  the  analysis  of  drinking 
water.  Now  that  analyses  of  drinking  water  are 
frequent,  Dr.  Miquel  offers  the  following  method, 
remarkable  for  the  simplicity  of  its  manipulations. 
In  a  small  precipitate  glass  with  a  foot,  and  closed 
by  an  emery-ground  tabulated  cap,  like  Pasteur's 
flasks,  a  little  wetted  cotton  is  placed.  Through 
the  tube  is  passed  a  platinum  wire,  crooked  at  the 
end  to  support  a  band  of  the  nutritive  paper  [F. 
45]  covered  on  both  sides,  about  3  cm.  wide  and 
8  long,  equivalent  to  about  half  a  decimetre  square 
surface;  all  is  sterilized  at  110°  C.  and  then 
weighed  to  a  milligramme.  The  mouth  is  now 
uncapped,  the  paper  plunged  for  one  or  two 
minutes  into  water  to  be  analyzed  for  the  different 
organisms,  then  introduced  into  the  eprouvette, 
and  re  weighed.  If  the  water  is  impure,  it  is  not 
long  before  small  spots  show  the  deposition  and 
growth  of  the  microbes,  which  spots  can  be  counted  ; 
and,  knowing  the  weight  of  water,  the  impurity 
can  be  rated  in  terms  of  comparison  according  to 
the  time  of  immersion, — the  increase  in  weight 
sometimes  exceeding  2  gr.  The  various  Bacteria, 
in  their  development  on  the  nutritive  paper,  offer 
different  appearances  even  to  the  naked  eye.  Pure 
cultures  can  thus  also  be  obtained.  In  any  case, 


BACTERIA    INVESTIGATION.  11 

when  the  germs  are  fully  developed,  the  paper  is 
again  dried  at  30-40°  C.,  by  which  the  colonies  are 
fixed,  and  they  can  be  preserved,  photographed,  or 
revived  if  the  paper  is  varnished  with  a  solution  of 
gum.  If  a  resin  varnish  is  used,  the  microbes  can- 
not be  revived. 

Miguel's  Method  for  ascertaining  the  hourly 
variations  of  aerial  Bacteria.  Dr.  Miquel  had  by 
previous  methods  shown  the  influence  of  rain,  of 
aridity,  of  humidity,  and  of  the  force  and  direc- 
tion of  the  wind,  upon  the  quantity  of  germs  held 
in  suspension  in  the  air ;  the  annual,  monthly, 
weekly,  and  daily  variations  in  the  quantity  of 
these  microscopic  organisms.  One  more  interest- 
ing point  remained  to  be  seen  ;  i.  e.,  whether,  in  the 
same  day,  —  the  meteorological  conditions  remain- 
ing sufficiently  fixed, — the  numbers  of  Bacteria 
vary  from  hour  to  hour,  like,  for  example, the  ther- 
mometric  pressure,  the  temperature,  etc.  Experi- 
ments described  in  the  Annuaire  de  Montsouris 
firmly  established  the  fact  that  at  noon  the  number 
of  Bacteria  were  twenty  times  less  than  at  eight 
o'clock  in  the  evening.  It  was,  however,  impor- 
tant to  know  whether  these  fluctuations  occurred 
with  regularity,  and  whether  they  could  be  de- 
tected every  day  at  the  same  hour,  providing,  of 
course,  they  were  not  interrupted  by  sudden 
and  unexpected  meteoric  phenomena.  Again,  ex- 
periments demonstrated  that,  under  normal  condi- 


12  THE  TECHNOLOGY   OF 

tions,  the  variation  of  aerial  Bacteria  is  effected 
with  regularity.  At  eight  o'clock  in  the  morning, 
the  number  of  atmospheric  Bacteria  is  always 
high;  after  this  time,  it  decreases  until  noon. 
From  noon  to  one  o'clock  it  is  at  a  remarkable 
minimum,  after  which  the  numbers  gradually 
increase  again  until  eight  in  the  evening.  Experi- 
ments made  at  night  gave  identical  results.  At 
ten  and  eleven  in  the  evening  the  air  is  very  im- 
pure ;  from  one  to  three  in  the  morning  it  has 
purified  itself  considerably,  to  again  have  the 
number  of  microbes  increase  in  the  forenoon .  The 
law  concerning  diurnal  variation  is  true  for  the 
entire  season.  Diverse  directions  of  the  wind  do 
not  modify  it,  providing  the  directions  remain 
constant.  As  to  the  force  of  the  wind,  one  is 
astonished  to  find  it  without  effect  upon  the  phe- 
nomena of  periodical  increase  and  decrease. 
Contrary  to  the  opinion  held  by  many,  the  air  is 
less  pure  morning  and  evening  than  at  mid-day. 
In  the  present  state  of  our  knowledge,  it  is  diffi- 
cult to  discern  the  cause  of  these  regular  variations. 
Dr.  Miquel  thinks,  however,  that  the  oblique 
currents  determined  on  the  surface  of  the  earth  by 
the  heating  and  cooling  of  the  soil  have  great 
effect  in  these  phenomena.  Those  winds  which 
sweep  along  the  surface  of  the  earth  naturally 
charge  themselves  with  a  larger  quantity  of  germs 
than  those  which  arrive  at  the  point  of  observation 


BACTERIA    INVESTIGATION.  13 

at  an  angle  of  incidence  of  80°  to  70°.  Dr.  Miquel 
regards  the  variable  obliquity  of  the  atmospheric 
currents  as  the  cause  of  the  increase  and  decrease 
of  microbes.  Unfortunately  meteorology  is  dumb 
as  to  the  nature,  frequence,  force,  and  periodicity 
of  these  oblique  movements  of  the  atmosphere. 
An  anemometer  should  be  invented  having  the 
faculty  of  measuring  the  inclination  of  the  wind. 
Dr.  Miquel  has  constructed  a  register  of  atmos- 
pheric Bacteria,  which,  if  not  capable  of  fur- 
nishing the  exact  number  of  germs,  is  capable 
of  showing  very  closely  their  variations.  The 
hourly  statistics  of  Bacteria,  by  the  process  of 
enumeration  employed  heretofore,  to  be  sufficiently 
exact,  required  600-800  preparations  of  beef  broth 
in  twenty-four  hours,  besides  five  persons  to  carry 
on  the  experiments  uninterruptedly  day  and  night. 
In  confiding  to  a  faithful  register  a  work  so  ex- 
pensive and  fatiguing,  a  step  in  advance  is  realized 
which  may  be  compared  to  the  progress  made  in 
meteorology  and  physiology  in  the  invention  of 
thermographs,  sphygmographs,  etc.  Some  two 
years  ago,  Dr.  Miquel  described  a  Mucedinograph 
or  Sporograph,  which  actually  figured  at  the  In- 
ternational Exposition  of  Hygiene  at  London,  in 
the  exceedingly  instructive  rooms  at  South  Ken- 
sington, and  by  the  side  of  which  was  to  be  seen 
a  Bacteriometer  which  is  far  from  being  so  simple 
an  affair,  and  of  which  the  following  is  a  descrip- 


14  THE  TECHNOLOGY  OF 

tion.  Apparatus. — Dr.  Miquel  adapted  to  a  clock- 
work made  by  MM.  Richard  freres,  a  plated  cop- 
per cylinder,  or,  better  still,  a  cylinder  of  ebonite, 
upon  the  exterior  of  which  was  placed  a  band  of 
nutritive  paper  [F.  45.]  10  cc.  wide  and  60  cc. 
long.  The  whole  is  placed  under  a  large  bell- 
glass  having  a  tubular  opening  through  the  knob 
on  its  summit  (through  which  aspiration  is  carried 
on).  The  borders  of  the  bell-glass  are  placed  in 
a  groove  made  in  a  plate  and  filled  with  mercury. 
A  vertical  slit  in  the  upright  portion  of  the  bell- 
glass  allows  the  projection  of  a  very  fine  jet  of  air 
upon  the  band  of  nutritive  paper,  which  makes 
one  revolution  in  twenty-four  hours,  and  receives 
the  microbes  in  the  atmospheric  dust  at  every 
instant  of  the  day.  Course  of  the  experiment.  1st. 
The  ebonite  cylinder  furnished  with  its  nutritive 
paper  is  sterilized  by  exposure  to  a  temperature 
of  110°  C.  for  one  hour.  2d.  The  cylinder  is 
then  placed  upon  the  clockwork,  and  beneath  the 
bell-glass,  which  is  coated  with  vaseline.  The  time 
of  starting  is  noted  and  a  constant  current  of  air 
is  determined,  varying  from  30  to  60  litres  per 
hour,  according  to  circumstances.  Twenty-four 
hours  afterwards  the  aspiration  is  suspended,  and 
the  results  noted  by  further  treatment  of  the  paper. 
3d.  Remove  the  ebonite  cylinder  with  its  band  of 
paper,  and  place  it  in  a  second  vaselined  bell-glass, 
where  the  Bacteria  may  incubate  and  develop 


BACTERIA    INVESTIGATION.  15 

protected  from  all  dust.  Precautions.  Not  only 
the  plates,  the  bell-glass  and  the  clockwork  need 
to  be  carefully  coated  with  vaseline  with  a  brush, 
but  it  is  necessary  to  cover  the  mercury  with  a 
thin  layer  of  aseptic  glycerine.  Moreover  the  air 
of  the  two  bell-glasses  must  be  kept  constantly  sat- 
urated with  moisture ;  this  may  be  easily  accom- 
plished by  placing  in  a  little  glass  dish  under  each 
cylinder  a  sponge  soaked  with  a  saturated  sub- 
limate solution. 

Hesse  s  Method  for  the  quantitative  estimation  of 
micro-organisms  contained  in  the  air  is  to  draw  the 
air  through  long  glass  tubes  whose  walls  are  coated 
with  stiffened  Koch's  culture  gelatine.  The  air 
stream  is  regulated  by  means  of  an  aspirator,  and  at 
the  same  time  measured.  The  number  of  the  colo- 
nies springing  up  in  the  gelatine,  and  the  quantity 
of  air  aspirated,  give  approximate  figures  for  the 
quantity  of  germs  in  the  air.  Hesse's  apparatus 
consists  of  a  glass  tube  (as  a  rule  90  cm.  long 
and  3.5  cm.  in  diameter),  closed  at  one  end  by  two 
rubber  caps,  the  inside  one  having  a  round  hole 
about  1  cm.  in  diameter  cut  in  the  centre.  The 
other  end  has  a  perforated  rubber  plug  2  cm.  thick, 
through  which  passes  a  glass  tube  with  cotton  in 
both  ends.  The  apparatus  is  sterilized  by  steam 
at  100°  C.  for  two  hours,  and  is  then  coated  inside 
with  culture  gelatine,  which  is  spread  evenly  by 
proper  movement,  the  bulk  being  allowed  to  settle 


16  THE  TECHNOLOGY   OF 

on  the  lower  side,  the  tube  being  held  horizontally. 
It  is  allowed  to  cool,  and  the  tube  placed  on  a 
tripod  such  as  is  used  for  photography,  and  the 
small  glass  tube  passing  through  the  stopper  is 
connected,  by  means  of  a  rubber  tube,  with  an 
aspirator  made  with  two  one-litre  flasks  connected 
by  rubber  tubes,  and  hung  to  the  same  stand,  the 
upper  one  filled  with  water  acting  as  an  aspirator. 
When  all  is  ready,  remove  the  outer  rubber  cap 
at  the  end  of  the  large  tube  with  disinfected 
hands. 

MarchancTs  Method  for  the  examination  of  mi- 
crobes or  corpuscles  held  in  suspension  in  water. 
The  limpid  water  is  placed  in  a  crystal  flask  cov- 
ered with  black  paper,  through  which  are  two 
opposite  square  openings,  one  destined  for  the 
passage  of  the  luminous  fluid,  the  other  for  obser- 
vation. On  allowing  a  rav  of  li^ht  to  traverse  the 

C?  J  o 

water,  it  does  so  without  obstacles  if  it  is  optically 
pure  ;  but  if  it  has  particles  in  suspension,  each  one 
of  these  is  appreciable  to  sight ;  but  without  this 
device  they  remain  invisible.  This  method  is  not 
new,  being  that  used  by  Tyndall  for  testing  the 
optical  purity  of  air. 

Certes'  Method  for  ascertaining  the  presence  of 
micro-organisms  at  great  depths  in  the  ocean.  He 
employed  an  ingenious  device,  made  by  M.  Alf. 
Milne-Edwards,  by  means  of  which  he  could  lower 
small,  hermetically  sealed,  sterilized  flasks,  and 


BACTERIA    INVESTIGATION.  17 

open  them  at  any  desired  depth,  ascertaining  at 
the  same  time  the  temperature  of  the  water  at  that 
point.  The  water  obtained  in  this  way  was  added, 
with  all  the  precautions  recommended  by  M.  Pas- 
teur against  contamination  from  atmospheric 
germs,  to  culture  fluids  made  of  sterilized  sea- 
water  (120°-128°  C.)  either  alone  or  in  combina- 
tion with  mutton  or  chicken  broth,  sterilized  hay 
infusion,  milk,  or  albuminous  broths,  Raulin  and 
Cohn's  fluids,  etc.  ^Erobic  microbes  were  found 
in  abundance,  but  no  anaerobic. 


LITERATURE    OF   THE    INVESTIGATION    OF    THE    BAC- 
TERIA CONTAINED  IN  THE  AIR,  WATER,  OR  EARTH. 

BECHAMP  :  "  Du  r61e  et  de  Porigine  de  certaines 
microzymas."  Compt.  Rend.  XCII.  (1881), 
p.  1344-7. 

BRAUTLECHT  (J.)  :  (On  the  transmission  of 
Bacteria  from  the  soil  into  the  air).  Tagebl. 
deutsch.  Naturf.  Vers.  Eisenach.  1882.  cf.  Natur- 
forscher  (1883),  p.  156.  Journ.  Roy.  Micr.  Soc., 
Ser.  II.,  vol.  III.  p.  541. 

CASSE  (J.)  :  "Terrain  et  microbes."  8vo,  18pp. 
Bruxelles  (A.  Manceux),  1884. 

CERTES  (A.)  :  I.  "  Sur  la  culture  a  1'abri  des 
germes  atmospherique,  des  eaux  et  des  sediments 
d'apportis  par  les  expeditions  du  '  Travailleur '  et 
du  r  Talisman,'  1882-1883."  Compt.  Rend.  T.  98, 


18  THE  TECHNOLOGY   OF 

p.  670  (1884).  II.  "  Sur  1'analyse  micrographique 
des  eaux."  Compt.  Rend.  T.  XC.  p.  1435.  Sep. 
pub.  Paris,  1883.  (B.  Tignol.)  (cf.  "Note  sur 
une  methode  de  conservation  des  Infusoires."  Ibid, 
seance  du  3  Mars,  1879.) 

FODOR  :  "Ueber  atmosphserischen  Staub,  etc." 
Hygienische  Untersuchangen,  Braunschweig,  1881. 

GIACOSI  (P.)  :  (On  organic  particles  in  the  air 
of  mountains).  Atti  R.  Accad.  Sci.  Torino.  XVIII. 
(1883),  p.  263. 

GUNNING  :  I.  "  Werden  mit  der  Expirationshift 
Bakterien  aus  Korper  entfuhrt."  Klin.  Monats.  f. 
Augenheilk.  Bd.  XX.  Hft.  1.  II.  «  Beitrag  zur 
hygienischen  Untersuchung  des  Wassers."  Archiv 
fur  Hygiene.  Bd.  L,  1883,  p.  335. 

HANSEN  (E.  C.)  :  (On  microorganisms  of  the 
air).  Resume  du  compte  rendu  des  travaux  du 
laboratoire  de  Carlsberg.  42  fasc.  Copenhagne. 
p.  197-258.  cf.  Journal  de  Microgniphie,  1882, 
T.  VI.,  p.  411. 

HESSE  (W.)  :  "Ueber  quantitative  Bestimmung 
der  in  der  Luft  enthaltenen  Mikroorganismen." 
Im  kaiserlichen  Ges.,  1884.  Bd.  2,  Taf.  xi.-xiii. 
II.  "Weitere  Mittheilungen  iiber  Lufttiltration." 
Deutsche  med.  Wochenschr.  1884.  No.  51. 

JORGENSEN  (A.)  OG  HoYER  (H.)  :  " Om  Drik- 
kevandet  i  Kolding"  (Microscopical  investigation 
of  drinking  water).  Kopenhagen,  1883.  8vo, 
25  pp.,  with  plates  and  figures  in  text. 


BACTERIA    INVESTIGATION.  19 

KIDDER  (J.  K.)  :  "  Keport  on  the  examination 
of  the  external  air  of  Washington."  Extr.  Report 
of  Surgeon  General  of  the  Navy  for  1880.  Wash- 
ington, 1882.  24  pp.,  10  tables. 

KOCH  (R.) :  (Microorganisms  in  soils)  Bied. 
Centr.  1883,  p.  581.  2.  cf.  Journ.  Roy.  Mic.  Soc. 
(1884)  p.  428.  Journ.  Chem.  Soc.  XLYI.  (1884) 
p.  486. 

MACDONALD  (J.  D.)  :  "A  guide  to  the  micro- 
scopical examination  of  drinking  water.  With  an 
appendix  on  the  microscopical  examination  of  the 
air."  2  ed.  (1883).  83  pp.,  8vo,  25  tab.  Phila. 
(Blakiston,  Son  &  Co.) 

MADDOX  (R.  L.)  :  "  On  a  portable  form  of  aero- 
scope  and  aspirator."  Journ.  Roy.  Mic.  Soc.,  Ser. 
II.,  vol.  III.  P.  2.,  1883,  p.  308. 

MAGGI  (L.) :  SulF  esame  microscopico  di  alcune 
acque  potabile  della  citta  e  per  la  citta  di  Padova. 
Pavie,  1883.  (Succ.  Bizzoni)  ( ff  La  partie  ge*ne*rale 
constitue  un  veritable  traite  d'analyse  microsco- 
pique  des  eaux,  analyse  dans  laquelle  la  recherche 
des  micro-organismes,  bacteries  et  autres,  occupe 
une  place  importante.")  Journal  de  Micrographie. 
1883. 

MARCHAND  (E.) :  "  Sur  1'examen  des  corpuscles 
tenus  en  suspension  dans  1'eau."  Compt.  Rend. 
1883.  T.  97,  p.  49. 

MIQUEL  (P.)  :  I.  "  Des  bacte*ries  atmosphe*rique." 
Compt.  Rend.  T.  91,  1880.  II.  "Etude  gtSne'ral 


20  THE  TECHNOLOGY  OF 

sur  les  bacteries  d'  1'atmosphere."  L'Annuaire  de 
Montsouris,  1881.  III.  "Etude  sur  les  pouss. 
organised  de  1'atmosphere. "  Nouvelle  rect.  Betris- 
sonia,  III.  5 ;  also,  Revue  de  botanique  crypt. 
III.  2  et  3.  IV.  "Des  procede*s  employes  pour 
recolter  les  germes  aeriens  des  bacteries."  Chapter 
Y.  in  "  Les  Organismes  vivant  de  1'atmosphere," 
Paris,  1883.  VI.  "Sur  le  dosage  des  Bacteriens 
dans  les  poussieres  et  dans  le  sol."  Bull,  de  la 
Soc.  Bot.  de  France.  T.  XXVIII.  (1881).  VII. 
(Hourly  Variations  of  Aerial  Bacteria,  MiqueFs 
Nutritive  Paper).  La  Semaine  Medicale,  Nov.  6, 
1884. 

MIFLET  :  "  Ueber  die  in  der  Luft  suspendirten 
Bacterien."  Betrage  zur  Biol.  d.  Pf,  III.  1. 

N^EGELI  u.  BUCHNER  :  "  Der  Uebergang  von 
Spaltpilzen  in  die  Luft."  Ctbl.  f.  d.  med.  Wiss. 
1882.  No.  29.  Absts.  in  Dtsch.  med.  Wochen- 
schr.  1882,  p.  516. 

PASTEUR  (L.)  :  "  Memoire  sur  les  corpuscles 
organises  qui  existent  dans  1'atmosphere."  An- 
nales  de  Chem.  et  de  Phys.  1862,  Ser.  3,  t.  64. 
Also  Compt.  Rend.  T.  48,  1859,  p.  337  ;  T.  50, 
1860,  p,  849. 

PASTEUR  ET  JOUBERT  :  "  Sur  les  germes  des 
bacteries  en  suspension  dans  1'atmosphere  et  dans 
les  eaux."  Compt.  Rend.  T.  84,  p.  206  (1877). 

POHL  (A.)  :  "  Chemische  und  Bakteriologische 
Untersuchungen,  betrefFend  die  Wasserversorgung 


.V 

^r 

UNIVEBSITY 


BACTERIA 

St.  Petersburgs  nebst  einem  Beitrag  zur  Biologie 
der  Microorganismen."  Petersburger  med.  Woch- 
enschr.  1884,  No.  31  u.  33.  —  Journ.  Russian 
Chern.  Soc.  1884.—  Nature,  XXIX.  (1884),  p.  557. 

SCHRODER  u  VON  DUSCH  :  "  Ueber  Filtration 
der  Luft  in  Beziehung  auf  Faulniss  und  Gahrung. 
Annalen  der  Chemie  und  Pbarmacie."  1854.  Bd. 
89,  p.  232. 

SCHUTZ  :  "Ueber  das  Eindringe  von  Pilzspor- 
en  in  die  Athemungswege  und  die  dadurch  beding- 
ten  Erkrankungen  der  Lungen  und  iiber  den  Pilz 
des  Hiihnergrindes."  Mitt.  a.  d.  kais.  Gesund- 
heitsamt.  1884.  Bd.  2. 

TIEMANN  :  "  Untersuchung  des  Wassers  auf 
entwicklungsfahigeMikroorganismen."  Yerhandl. 
dtsch.  Gesellsch.  f.  offentl.,  Gesundheitspflege  zu 
Berlin,  1883. 

TISSANDIER  (G.)  :*  "  Les  poussieres  de  1'air." 
Paris  (Gauthier  et  Villars),  1877.  1  vol.,  18mo. 

TYNDALL  (J.)  :  "Essays  on  the  Floating  Matter 
of  the  air."  2d  edit,  1883. 

WERNICH  (A.):  I.  "Ueber  das  Haften  und 
die  Ansiedlungsfahigkeit  staubformige  Pilzkieme." 
Dtsch.  med.  Woehenschr,  1882,  p.  513.  II. 
"  Die  Luft  als  Tragerin  entwicklungsfahiger 
Keime."  Yirchow's  Archiv.  Bd.  79,  p.  424-445. 

WOLLNY  (E.)  :  "  Ueber  die  Tatigkeit  niedere 
Organismen  im  Bo  Jen  "  Deutsche  Vierteljahrsschr. 
f.  oeflfentl.  Gesundheitspflege,  XV.  4. 


22  THE  TECHNOLOGY   OF 

If  a  person  desires  to  isolate  for  investigation, 
from  some  fluid  containing  several  forms  of  Bac- 
teria, some  one  particular  form,  he  may  best  pro- 
ceed according  to  — 

JWoegeli's  Dilution  Method,  as  illustrated  in  the 
following  example.  Prof.  Naegeli  had  some  urine 
containing  large  Cocci  and  also  numerous  Bacilli ; 
the  former  he  desired  to  obtain  pure.  A  drop  of 
the  urine  (about  0.03  ccm.),  estimated  to  contain 
500,000  Bacteria,  was  placed  in  30  ccm.  of  pure 
sterilized  water;  after  being  thoroughly  shaken, 
one  drop  of  this  thousand  times  diluted  urine  was 
added  to  a  second  30  ccm.  of  water,  and  thus  a 
millionth  dilution  was  obtained,  in  which  every 
two  drops  of  0.03  ccm.  must  contain  one  microbe. 
Each  of  ten  tubes  of  sterilized  culture  fluid  [vid. 
CULTURE  METHODS]  was  then  inoculated  with  one 
drop  of  this  diluted  urine,  and  after  proper  incuba- 
tion it  was  found  that  four  tubes  remained  sterile, 
one  contained  Bacilli,  and  five  the  desired  Cocci. 
These  he  could  now  study  alive  as  microscopical 
preparations,  or  by  any  method  open  for  the  inves- 
tigation of  Bacteria.  —  On  the  other  hand,  the 
Bacteria  which  it  is  desired  to  study  may  not  be  in 
a  condition  suited  for  microscopic  study,  they  may 
be  dried  or  in  the  "resting  spore"  condition,  re- 
quiring special  treatment  before  they  can  be  stud- 
ied alive,  undergoing  the  various  phenomena  of 
their  life  history  ;  thus  — 


BACTERIA    INVESTIGATION.  23 

Ituchner's  and  Robert's  Method  for  Bacillus 
subtilis  (the  Hay  Bacterium)  is  to  digest  a  quan- 
tity of  finely-chopped  hay  for  an  hour,  with  as 
little  water  as  possible,  at  a  temperature  of  36°  C. 
The  liquid  is  then  drained  off  through  a  wire  sieve, 
and  diluted  with  distilled  water  until  it  reaches  a 
specific  gravity  of  1.004.  If  the  fluid  is  acid  in 
reaction,  it  must  now  be  neutralized  with  car- 
bonate of  soda,  and  not  less  than  500  ccm.  placed 
in  a  flask,  which  is  stopped  with  a  plug  of  cotton. 
The  flask  and  contents  are  now  retained  for  forty- 
eight  hours  at  a  temperature  of  36°  C.,  when  the 
developed  Bacilli  will  have  formed  a  dry-looking 
pellicle  upon  the  surface. 

BACTERIA  IN  HEALTHY,  LIVING  ORGANISMS. 

Peculiar  procedures  had  to  be  employed  for 
developing  the  bacterial  germs  which  exist  in  the 
tissues  of  healthy  living  animals,  and  rendering 
them  suitable  objects  for  microscopic  examination. 
The  first  of  these  was  — 

TiegeTs  Method.  The  organ  to  be  examined,  or 
a  piece  of  it,  is  removed  with  a  sterilized  knife  from 
an  animal  just  killed  (usually  by  bleeding  through 
the  carotid) .  A  silk  thread,  previously  well  boiled, 
is  quickly  tied  about  the  piece,  and  by  means  of 
this  it  is  dipped  in  melted  paraffins  at  110°  :  115°  C., 
and  allowed  to  remain  a  longer  or  shorter  time, 
according  to  the  size  of  the  piece.  After  the 


24  THE   TECHNOLOGY   OF 

paraffine  remaining  on  the  surface  lias  cooled,  it  is 
again  dipped  and  very  quickly  withdrawn.  This 
is  only  intended  for  the  purpose  of  strengthening 
the  paraffine  crust.  After  this  layer  has  cooled, 
the  preparation  is  dropped  into  a  mass  of  cooling 
paraffine  at  52°  C.,  and  here  allowed  to  cool.  The 
lump  thus  formed  is  then  for  a  length  of  time, 
4  to  12  days,  retained  at  a  temperature  of  about 
30°  C.,  then  cut  open  and  the  interior  studied. 
The  strong  scalding  which  the  organ  receives  in 
the  first  paraffine  kills  such  microbes  as  may  have 
fallen  upon  it  during  its  removal,  and  before  they 
can  have  reached  the  interior.  Billroth  and  Tie- 
gel  always  found  Bacteria  in  the  pancreas,  liver, 
spleen,  salivary  glands,  testicles,  and  muscles  after 
4  to  12  days  ;  most  in  pancreas. 

Bur  don  Sanderson's  Method,  to  overcome  the 
objection  that  the  Bacteria  might  have  gained 
entrance  to  the  tissue  through  the  cracks  and 
pores  formed  in  the  paraffine  while  cooling,  was 
to  throw  the  organ  into  melted  paraffine  at  110°  C., 
and,  as  soon  as  it  was  cool  and  stiff,  to  paint  it 
over  with  Venetian  turpentine.  Sometimes  he 
simply  dropped  the  organ  into  oil  at  110°  C.  and 
allowed  it  to  remain  at  the  bottom  of  the  vessel  for 
1  to  2  days.  The  surface  was  found  to  be  cooked, 
but  the  central  portion  retained  the  pale  red  color 
of  raw  tissue,  and  contained  numerous  Bacteria  at 
different  stages  of  development.  Richtet  employed 


BACTEKIA  INVESTIGATION.  25 

this  method  in  seeking  Bacteria  in  fish.  Thus,  in 
the  case  of  a  live  eel,  he  opened  it  with  sterilized 
scissors,  cut  out  its  liver,  and  plunged  this  into 
melted  paraffine  at  110°  C.  After  cooling,  the 
mass  was  covered  with  several  coats  of  collodion, 
and  then  with  Canada  balsam.  Three  weeks  after, 
the  liver  contained  myriads  of  small  Bacilli,  but 
it  still  had  the  odor  of  fresh  fish. 

Chiene  and  Eivarfs  Method :  Under  a  spray  of 
5  per  cent  carbolic  acid  solution,  the  body  of  a 
rabbit,  just  killed,  was  opened,  and  the  liver, 
spleen,  kidneys,  and  pancreas  taken  out  and  cut 
into  pieces.  Some  of  these  were  dipped  into  a 
solution  of  carbolic  acid,  others  were  wrapped  in 
antiseptic  gauze  ;  they  were  then  placed  in  vessels 
which  had  been  heated  red  hot,  and  the  openings 
closed  with  antiseptic  gauze,  cotton,  or  with  a  glass 
cover.  In  those  pieces  which  had  been  dipped 
into  the  antiseptic  solution  they  found  no  Bacteria, 
and  hence  concluded  that  there  were  none  present 
in  the  organs.  This  gave  rise  to  — 

Nencke  and  Giacosi's  Methods,  proving  that 
Chiene  and  Ewarts  had  killed  the  Bacteria  with 
carbolic  acid. 

(a)  In  a  beaker-glass  of  the  capacity  of  about 
one-half  litre,  a  quantity  of  some  alloy  which  is 
readily  melted,  say  at  75°  C.  [Wood's  metal],  is 
heated  up  to  300°  or  400°  C.  As  soon  as  the 
alloy  has  cooled  to  150°  C.,  a  layer  of  5  per  cent 


26  THE   TECHNOLOGY   OF 

carbolic  acid  solution  is  poured  upon  it,  and  the 
metal  is  retained  in  a  melted  condition  by  being 
placed  in  a  water-bath.  Now,  a  rabbit  is  killed, 
and  its  abdomen  shaved  and  washed  with  the  car- 
bolic acid  solution.  The  abdominal  cavity  is  then 
opened  under  a  spray  of  5  per  cent  carbolic  acid 
solution,  and  a  large  piece  of  the  liver  cut  out  and 
placed  with  sterilized  pincers  in  the  melted  Wood's 
metal,  and  the  whole  allowed  to  cool  and  stiffen 
about  both  tissue  and  pincers.  The  mass  is  then 
kept  for  four  days  at  40°  C.  In  this  method  there 
is  no  danger  of  the  material  enclosing  the  tissue 
cracking,  and,  if  this  should  happen,  no  germs 
from  the  air  could  reach  the  enclosed  tissue  on 
account  of.  the  intervening  5  per  cent  carbolic  acid 
solution. 

(6)  Nencki  and  Giacosi's  second  method  is  to 
take  a  large  enamelled  iron  vessel,  and  fill  it  one- 
third  full  of  pure  mercury.  A  glass  tube  about 
5  ctms.  in  diameter  is  taken,  and  one  end  melted 
round,  like  the  fundus  of  a  test  tube,  and  the  open 
end  ground  perfectly  even.  This  is  then  filled 
with  mercury,  closed  with  a  glass  plate,  and  in- 
verted into  the  vessel,  being  allowed  to  remain 
leaning  against  the  side.  The  whole  apparatus  is 
then  heated  until  the  dish  is  about  two-thirds  full 
of  mercury.  Any  germs  which  might  be  in  the 
tube  are  killed  by  the  boiling  mercury.  It  is  now 
allowed  to  cool,  whereby  the  mercury  is  again 


BACTERIA    INVESTIGATION.  27 

condensed  in  the  tube,  and,  when  the  temperature 
reaches  the  neighborhood  of  120°,  a  5  per  cent  solu- 
tion of  carbolic  acid  is  poured  over  the  surface. 
With  proper  precautions  and  the  use  of  the  carbo- 
lized  spray,  some  organ  of  a  freshly  killed  rabbit 
(liver,  heart,  kidney,  spleen,  pancreas)  is  taken  and 
placed  with  the  pincers  under  the  mouth  of  the 
tube,  to  the  top  of  which  it  rises,  and  there  re- 
mains. The  preparation  is  now  kept  at  a  tem- 
perature of  40°  C.  for  one  or  more  days.  In  these 
methods  all  organs  investigated  showed  the  pre- 
sence, of  Bacteria  in  large  numbers. 

LITERATURE    ON    THE    PRESENCE    OF    BACTERIA    IN 
HEALTHY   LIVING    ANIMALS. 

BABES  (V.)  :  (No  Bacteria  in  blood  or  tissues 
of  healthy  men).  Biol.  CentralbL,  1882,  II.,  p. 
97-101. 

BALZER  (Leptothrix  epidermadis) :  Annales  de 
Dermat  et  de  Syphilographie,  25  Dec.,  1883,  p. 
681. 

BECHAMPS  (J.)  :  I.  "  Les  Microzymas  "  (Micro- 
cocci).  Montpellier  et  Paris,  1875.  II.  "Des 
microzymas  gastriques,  et  de  leur  pouvoir  diges- 
tif." Compt.  Eend.  27  Feb.,  1882.  (Journal  de 
Micrographie,  T.  IV.,  p.  188,  1882. 

BECHAMPS  (A.),  ESTOR  (A.),  ET  SAINTPIERRE 
(C.)  :  "  Du  r61e  des  organismes  microscopiques  de 
la  bouche  dans  la  digestion  en  ge*ne*ral,  et  particu- 


28  THE  TECHNOLOGY  OF 

lierement    dans    la    formation    de    la    diastase." 
Compt.  Eend.  T.  LXIV.,  p.  696  (1867). 

BEIMSTOCK  (B.)  :  "  Ueber  die  Bakterien  der 
Faeces,"  Fortschritte  der  Med.,  1883,  No.  19; 
also  Zeitschr.  f.  klin.  Med.  VIII. 

BILLROTH  und  TIEGEL  :  "  Ueber  Cocco  Bacteria 
septica  (Billroth)  im  gesunden  Wirbelthierk  rper." 
Virchow's  Archiv.  f.  pathol.  Anat.  u.  s.  w.  Vol.  60, 
p.  453. 

BIZZOZERO  (G.):  "Sui  Microfiti  dell' Epider- 
mide  umana  normale,"  Estratto  dal  volume  d'Atti. 
della  E.  Accad.  di  medicina  di  Torrino,  1884.  Cf. 
Virchow's  Archiv.,  1884,  Bd.  98,  p.  441. 

BURDON-SANDERSON  :  "  Bacteria  in  healthy  living 
organs."  British  Med.  Journ.,  Jan.  26,  1878. 

CARL  (Herzog  v.  Bayern)  :  "  Bei  Menschen 
vorkommende  Bacillen  "  (in  the  choroid).  Abth. 
f.  med.  Wiss.,  1881,  I.  Louisville  Med.  Herald, 
iii.  (1881),  p.  27,  Journ.  K.  Mic.  Soc.,  Ser.  II. 
vol.  vi.,  p.  644. 

CHIENE  (J.)  and  EWART  (C)  :  "Do  bacteria  or 
their  germs  exist  in  the  organs  of  healthy  living 
animals."  Journ.  of  Anat.  and  Physiol.,  vol.  xiii. 
part  3,  p.  448,  1878. 

DOWDESWELL  (G.  F.)  :  "  On  some  appearances 
in  the  blood  of  vertebrated  animals,  with  reference 
to  the  occurrence  of  Bacteria  therein."  Jour.  Roy. 
Micr.  Soc.,  1884,  p.  525-529. 

ESTOR  (A.)  :    "  Contribution  a  1'etude  des  mi- 


BACTERIA  INVESTIGATION.  29 

crozymas  et  des  bacteries."  8vo,  20  pp.  Paris 
(Delahaye  et  Lecrossier),  1884. 

FRISCH  (A.)  :  "  Experimentelle  Studien  lib.  d. 
Yerbreitg.  d.  Faulnissorganismen  in  d.  Geweben." 
mit.  5  Tafln,  4to,  1874. 

HOFFMAN  (G.  v.) :  "  Untersuchungen  iiber 
Spaltpilze  im  menschlichen  Blute,  Ein  Beitrag  zur 
allgemeinen  Pathologic."  Gr.  8,  mit  2  lith.  Tail. 
1884. 

KUNSTLER  (J.)  :  (Bacterioidomonas  sporifera 
in  caecum  of  Cavia).  Journal  de  Micrographie, 
1884,  VIII.,  pp.  376-80.  Jour.  Koy.  Mic.  Soc., 
1884,  p.  934. 

LEUBE  (W.  O.)  :  "Beitrag  zur  Frage  von  Vor- 
kommen  der  Bakterien  in  lebender  Organismus, 
speciell  im  frischgelassenen  Harn."  Zeitschr.  f. 
klin.  Med.  III.,  p.  233. 

LEWIS  (T.  E.)  :  I.  "Microscopic  Organisms 
found  in  the  Blood  of  Man  and  Animals."  Four- 
teenth Annual  Eeport  of  the  Sanitary  Commis- 
sions with  the  Government  of  India.  II.  "Mi- 
crophytes in  the  Blood,  and  their  Relation  to 
Disease."  Quart.  Journ.  Mic.  Sci.,  XIX.  (1879;, 
p.  356.  — Journ.  Eoy.  Mic.  Soc.,  1879,  p.  924. 

LUDWIG  (F.)  :  I.  (Micrococcus  prodigiosus 
within  the  shell  of  an  egg.)  Zeitschrift  f.  Pilz- 
freunde,  1883,  p.  176.— Botan.  Ctbl.  Ixviii.  (1884), 
p.  160.  Journ.  Eoy.  Mic.  Soc.  (1884),  p.  596. 
II.  (Photogenous  Micrococci,  Micrococci  pflugeri 


30  THE   TECHNOLOGY   OF 

the  cause  of  phosphorescence  in  fish,  Crustacea, 
echinoderms,  etc.,  and  of  phosphorescent  patches 
on  the  surface  of  the  sea.)  Hedwigia,  XXII. 
(1884),  p.  106-15.  Journ.  Eoy.  Mic.  Soc.,  1884, 
p.  596. 

METSCHNIKOFF  (E.) :  "  Untersuchungen  iiber 
die  Intracellulare  Verdauung  bei  Wirbellosen 
Thieren  "  (Bacteria  in  Tunicates  fresh  from  the  sea, 
p.  21),  Wien,  1883.  (Transl.  in  Quart.  Journ. 
Micros.  Sci.  for  Jan.,  1884.)  II.  "Ueber  die 
pathologische  Bedeutung  der  intracellularen  Ver- 
dauung," Virchow's  Archiv,  Bd.  XCVL,  Hft.  2, 
1884.  cf.  Arbeiten  des  zoolog.  Instit.  zu  Wien, 
Bd.  V.,  Hft.  2,  1883.  — Biologisches  Centralbl. 
Bd.  III.  No.  18.  1883. 

MEYER  (L.)  :  (On  rod  Lke  bacteria  found  on 
the  mucous  surfaces  of  the  female  sexual  organs). 
Vortrag  in  der  Ges.  fur  Geburtshilfe,  Berlin, 
1862. 

MILLER  (W.  D.)  :  "  Zur  Kenntniss  der  Bakter- 
ien  in  der  Mundhohle,"  in  Deutsche  med.  Woch- 
enschr.  No.  48,  1884.  p.  781. 

Moss  (E.  L.)  :  "  On  septic  organisms  in  living 
tissues."  Journ.  Koy.  Mic.  Soc,  (1879),  p.  312. 

NENCKI  (M.)   UND  GIACOSA   (P.)  :  "  Giebt  es 
Bacterien  und  deren   Keime  in  den  Organer  ge- 
sunder  lebener  Thiere,"  in  Beitrage  zur  Biologie 
der  Spaltpilze,  Leipzig,  1880. 
.     NOTHNAGEL  (H.)  i  "  Bacillus  Amylobacter  (Clo- 


BACTERIA  INVESTIGATION.  31 

stridium  butyricum)  im  Darwinhalt."  Ctbl.  f.  d. 
med.  Wiss.,  1881,  p.  19. 

PETER.  (Bacteria  as  the  cause  of  the  various 
coloring  of  the  eggs  of  Coregonus  ivartmanni.) 
Berl.  bot.  Yerein,  Miinchen,  Sept.  19,  1883. 
Journ.  Eoy.  Micr.  Soc.,  1884,  p.  601. 

RASMUSSEN  :  "  Om  Drychning  af  Mikroorganis- 
merfra  Spyt  af  Sunde  Mennesker  "  (On  the  culture 
of  the  micro-organisms  of  the  saliva  of  healthy 
men).  Kopenhagen,  1883.  Dissertation. 

REYMOND  :  "  Batteri  del  sebo  delle  ghiandole 
meibomiane  normale."  Giornale  dell'  Ace.  di 
Med.  di  Torino.  (Lugli.),  1883. 

RICHET  (Ch.) :  "  Les  microbes  des  poissons 
marins."  Comptes  Rendus  de  la  Acad.  des  Sciences 
de  Paris.  Tome  96,  p.  384  (1883). 

ROBERTS  0V.)  :  ffOn  the  occurrence  of  micro- 
organisms in  fresh  urine."  Brit.  Med.  Journ., 
1881,  Oct.  15. 

ROCHAS  (F.)  :  "Les  schizophytes  parasite  de 
rhomme  et  des  animaux."  Lyon.  (Georg),  1885, 
pp.  27,  8vo. 

ROSBACH  (M.  J.)  :  "Ueber  die  Vermehrung  der 
Bakterien  in  lebenden  Thiere  nach  einverleibung 
eines  chemischen  organismen  freien  Fermentes." 
Ctbl.  f.  d.  med.  Wiss.,  1882,  No.  5. 

STERNBERG  (G.  M.)  :  "  A  contribution  to  the 
study  of  the  bacterial  organisms  commonly  found 
upon  exposed  mucous  surfaces  and  in  the  alimen- 


32  THE  TECHNOLOGY  OF 

tary  canal  of  healthy  individuals."  Studies  from 
the  Biol.  Lab.  Johns  Hopkins  University.  II. 
(1882),  pp.  157-181. 

TRINCHESE  :  (Bacteria  in  the  Human  Amnion.) 
Atti  K.  Acad.  Lincei.  1884,  p.  237.  Journ. 
Eoy.  Micros.  Soc.  (1884),  p.  268. 

VULPIAN:  Bull,  de  1'Acad.,  29  Mars,  1881. 

WASSILIEF  (N.  P.) :  "  Beitrag  zur  Frage  iiber 
die  Bedingungen  unter  denen  es  zur  Entwicklung 
von  Mikrokokken  Colonien  in  den  Blutgefassen 
kommt."  Ctbl.  f.  d.  med.  Wiss.  1881.  No.  52. 

ZAHN  :  "  Untersuchungen  iiber  das  Vorkommen 
von  Faulnisskeimen  im  Blut  gesunder  Thiere." 
Archiv.  f.  pathol.  Anat.  1884.  Bd.  CXXXV., 
p.  401. 

To  obtain  blood  for  microscopical  examination, 
remove  the  hair  from  the  spot  to  be  punctured, 
clean  the  skin  carefully  with  warm  water  and  soap, 
place  a  drop  of  0.8  per  cent  salt  solution  on  the 
spot,  and  with  a  knife,  which  has  been  previously 
heated,  cut  through  the  skin ;  then  with  another 
knife  cut  a  small  vein  or  artery. 

Moss's  Method  for  obtaining  blood  for  bacte- 
riological investigation.  This  method  seems  to 

c?  O 

exclude  all  possibility  of  infection,  and  at  the 
same  time  allows  the  blood  to  be  examined  at 
intervals.  The  apparatus  consists  of  a  series  of 
small  glass  bulbs  connected  together  by  capillary 
tubes,  so  that  one  bulb  and  its  contents  can  be 


BACTERIA    INVESTIGATION.  33 

separated  from  the  rest  by  the  blow  pipe.  The 
tubes  and  bulbs  are  bent  on  each  other,  so  that  the 
whole  series  can  be  readily  sterilized  in  a  water 
or  paraffin  bath.  One  end  of  the  series  is  left 
open,  packed  with  sterilized  cotton,  and  connected 
with  an  aspirator,  the  other  drawn  to  a  fine  point 
and  sealed.  The  sealed  point  is  secured  in  a  stout 
piece  of  india-rubber  connection  pipe,  which  is 
fastened  over  the  collar  of  a  fine  hypodermic 
needle,  protected  ready  for  use  in  a  calcined  glass 
sheath.  The  whole  arrangement  is  then  repeatedly 
baked  in  a  water  bath  at  intervals  of  four  hours. 
(TyndaPs  method  of  sterilization  by  discontinuous 
heating.)  In  using  the  apparatus  the  sheath  is 
removed  from  the  needle,  and  the  latter  plunged 
into  any  suitable  vein.  The  sealed  point  inside 
the  rubber  connection-tube  is  broken,  and  the 
blood  flows  gently  through  the  series  of  bulbs, 
drawn  on  by  the  aspirator  acting  through  the  cotton 
plug.  When  sufficient  has  entered,  the  blow-pipe 
severs  the  tube  next  the  needle,  and  instantly  after- 
wards that  next  the  cotton  plug. 

Only  in  two  diseases  are  Bacteria  regularly  to  be 
found  in  the  blood  of  living  persons,  i.  e.,  in  An- 
thrax and  Febris  recurrens.  The  anthrax  Hacitti 
withstand  the  action  of  most  reagents,  while  the 
Spirochcete  of  recurrent  fever  are  readily  destroyed 
by  the  addition  of  fluids,  even  by  distilled  water. 

To  obtain  serum  or  fluid  from  a  freshly  extracted 


34  THE   TECHNOLOGY   OF 

organ,  cut  out  a  piece  with  a  previously  heated 
knife,  and  with  another  sterilized  knife  cut  through 
this  piece,  and  press  a  clean  cover-glass  against  the 
freshly  cut  surface ;  or  by  scraping  the  surface 
with  the  sterilized  platinum  wire,  sufficient  fluid 
may  be  obtained,  and  transferred  to  the  cover- 
glass.  If  the  cut  surface  of  the  organ  or  tissue 
be  of  a  consistency  not  easily  spread  out,  a  few 
drops  of  sterilized  water  should  be  placed  upon 
the  freshly  cut  surface  before  laying  the  cover-glass 
upon  it  or  scraping  it  with  the  wire. 

The  Study  of  Microscopical  Preparations  of 
fixed  and  Hardened  Bacteria,  while  not  by  any 
means  the  most  important,  is  the  method  which 
has  been  most  largely  employed.  When  the  Bac- 
teria are  to  be  examined  in  opaque  substances, 
such  as  fresh  tissues,  aiucus,  or  fluids  containing 
much  detritus,  they  must  be  rendered  visible  upon 
the  slide  by  means  of  clearing  agents.  Fresh 
tissues  (tissues  which  have  been  for  a  length  of 
time  in  alcohol  or  other  hardening  fluids  are  not 
readily  cleared  up,  and  therefore  not  easily  exam- 
ined without  staining)  should  be  examined  as  early 
as  possible  after  removal,  in  sections  cut  with  a 
freezing  microtome  or  a  Valentin's  knife ;  these, 
as  well  as  turbid  fluids,  should  be  spread  out  upon 
the  slide,  and  cleared  up  with  either  a  15  per  cent 
sulphuric  acid  solution,  a  20  per  cent  solution  of 
caustic  potash,  concentrated  acetic  acid,  absolute 


BACTERIA    INVESTIGATION.  35 

alcohol,  or  ether,  as  may  seem  proper  for  the  case 
in  hand.  Usually  the  microbes  withstand  the 
action  of  the  clearing  fluids,  while  the  tissue  or 
detritus  almost  entirely  disappears.  Examine 
these  preparations  with  a  narrow  light.  It  is 
not  infrequently  the  case  that  sections  of  tissues 
from  cases  of  metastatic  pysemia,  endocarditis 
ulcerosa,  and  acute  croupous  pneumonia  show  the 
blood-vessels,  capillaries,  small  veins,  and,  in  the 
last-named  disease,  the  lymph  vessels,  to  be  irreg- 
ularly filled  with  heaps  or  chains  of  a  granular 
material.  These  "  capillary  emboli  "  may  consist 
of  Micrococci  colonies,  which  may  be  considered 
proven  if  we  find  that  they  withstand  the  action  of 
clearing  agents,  as  indicated  above.  Control  tests 
should  be  made  by  staining  some  sections. 

The  Use  of  Staining  Fluids,  in  connection  with 
Bacteria  which  have  not  been  hardened  or  fixed, 
is  not  satisfactory  ;  and  yet  they  are  often  still  less 
easily  stained  after  having  been  for  a  long  time 
exposed  to  the  action  of  alcohol  or  other  harden- 
ing agents,  or  after  having  been  for  a  long  time 
dried.  The  best  results  are  to  be  obtained  from 
tissues  which  have  remained  for  only  a  short  time 
in  hardening  fluids.  Staining  fluids  are  employed 
either  for  the  so-called  "  cover-glass  preparations," 
in  which  the  Bacteria  to  be  studied  exist  in  fluids, 
or  for  the  preparation  of  sections  in  cases  where 
the  substratum  is  a  firm  one.  Bacteria  behave  in 


36  THE  TECHNOLOGY  OF 

staining  much  like  nuclei,  and,  as  a  rule,  such 
staining  fluids  as  are  good  for  nuclei  will  succeed 
with  Bacteria.  On  account  of  their  minute  size, 
however,  the  more  intense  the  stain,  the  better ; 
therefore  the  aniline  dyes  seem  best  suited.  The 
intensity  of  the  staining  can  be  increased  by  keep- 
ing the  preparations  at  a  temperature  of  about 
50°  C.,  in  a  drying-oven.  The  behavior  of  Bac- 
teria and  nuclei  is  very  different  towards  alkalies  ; 
the  nuclei  are  dissolved  by  alkalies,  while  Bacteria 
are  unaffected ;  therefore  the  staining  fluids  are 
often  made  alkaline  in  order  that  any  confusion 
of  Bacteria  with  nuclei  may  be  avoided.  For 
Micrococci  most  nuclear  stainings  may  be  used. 
Koch's  and  Gram's  general  methods,  or  Lceffler's 
method  given  under  diphtheria,  give  good  results. 
It  is  known,  however,  that  living  Micrococci  take 
staining  much  better  than  do  dead  ones.  It  is 

o 

possible  that  further  study  of  the  way  in  which 
different  species  of  Micrococci  take  staining  may 
show  characteristic  specific  peculiarities,  but  as 
yet  there  are  none  known  ;  the  Micrococci  of  malig- 
nant endocarditis,  of  pyaemia,  of  erysipelas,  and 
of  gonorrhoaa,  etc.,  behave  alike.  Weigert  rec- 
ommends, for  red,  all  carmines  (Schvveigger-Sei- 
del's),  purpurin,  fuchsin,  or  Magdala-red,  for 
brown,  Bismarck-brown  or  vesuvin ;  for  brown- 
violet,  carmine,  followed  by  washing  in  alcohol  to 
which  has  been  added  a  little  liquor  ferri  sesqui- 


BACTERIA    INVESTIGATION.  37 

chloride ;  for  green,  methyl-green ;  for  blue  and 
violet,  hsematoxylin,  iodine-violet,  methyl-violet, 
gentian-violet,  or  dahlia.  He  recommends  for 
Bacilli  the  nuclear  staining  aniline  dyes,  those,  for 
instance,  which  Ehrlich  designates  "  basic  ani- 
lines,"—  Bismarck-brown,  methyl-violet,  methyl- 
green,  saffranin,  fuchsin,  Magdala-red,  etc. 
Excelling  all  these,  however,  for  Bacilli,  is  gen- 
tian-violet. Use  all  the  anilines  in  strong  aque- 
ous solutions  until  the  preparation  is  over-stained, 
and  remove  the  excess  by  washing  in  acetic  acid, 
water,  or  in  alcohol.  Carmine  and  heematoxylin 
are  not  recommended  for  Bacilli,  although  Klebs 
used  the  latter  with  good  success  in  his  researches 
regarding  the  Bacillus  of  typhoid  fever. 

Hazlewood  recommends,  as  a  general  blue  stain, 
a  mixture  of  rosanilin,  aniline  oil,  and  sulphuric 
acid,  as  giving  "  surprisingly  fine  results  with 
Micrococci,  Bacilli,  etc.  Whether  the  mount  is 
in  balsam  or  glycerine,  the  result  is  excellent, 
though  the  glycerine  mounts  are  in  some  respects 
preferable." 

Blanchard's  Method:  In  a  few  hours,  or  two 
days  at  the  longest,  the  surface  of  water  in  which 
an  organized  substance  (vegetable  or  animal  tissue, 
etc.)  has  been  macerated,  becomes,  as  is  well 
known,  covered  with  a  slight  pellicle,  composed  of 
a  more  or  less  compact  mass  of  Bacteria,  envel- 
oped in  a  hyaline,  transparent  substance  of  slight 


38  THE  TECHNOLOGY  OF 

consistence.  A  tolerable  large  piece  of  this  mem- 
brane ("  proliferous  membrane  "  Pouchet)  can  be 
obtained  by  introducing  into  the  fluid  beneath  it 
a  glass  slide,  and  raising  it  with  caution.  The 
film  thus  obtained  is  fixed  by  treating  it  with 
strong  osmic  acid.  It  is  then  covered,  the  osinic 
acid  drawn  off.  and  a  drop  of  methyl-violet  run  in 
under  the  cover.  In  half  ai>  hour's  time  the 
preparation  may  be  completed  by  running  in  gly- 
cerine, to  which  is  added  a  small  quantity  of  the 
violet,  in  order  that  the  stain  may  not  be  extracted 
from  the  organisms ;  or  a  concentrated  solution 
of  sulphate  of  calcium  may  be  used  instead  of 
glycerine.  The  Bacteria  are  stained  a  fine  violet, 
the  ground  substance  remaining  colorless.  Other 
aniline  dyes  may  be  used,  but  methyl-violet  seems 
to  be  the  most  durable.  Hsematoxylin  may  also  be 
used.  In  this  case,  the  film  of  Bacteria  should  be 
stained  in  it  for  twenty-four  hours  (after  fixing  with 
osmic  acid)  ;  the  iridescence  which  is  then  formed, 
and  which  spoils  the  clearness  of  the  preparation,  is 
removed  by  repeated  washing,  and  the  membrane 
is  mounted  in  glycerine,  or  chloride  of  calcium. 

"  T.  C"1  recommends  the  following  method  of 
obtaining  permanent  preparations  of  Bacteria,  it 
being  the  result  "  of  some  years  of  patient  re- 
search." A  ring  of  white  wax  much  larger  than 

i  Science  Gossip,  1879.    No.  173,  p.  111. 


BACTERIA    INVESTIGATION.  39 

the  cover-glass  is  drawn  upon  a  slide,  within 
which  the  solution  containing  the  microbes  is 
placed.  When  they  have  attached  themselves  to 
the  slide,  some  of  the  fixing  solution  (25  cc.  of 
chromic  oxi-chloride  acid,  to  which  is  added  50  cc. 
of  water  with  50  cc.  of  permanganate  of  potash) 
is  added,  which  will  instantly  kill  and  fix  the  speci- 
mens ;  after  three  minutes,  the  water  may  be 
poured  out,  a  few  drops  of  absolute  alcohol  and 
then  of  chloroform  added  and  poured  off,  the 
cover-glass  placed  carefully  on,  and  some  thin 
Canada  balsam  run  under. 

It  is  important,  in  the  use  of  aniline  dyes,  that  the 
materials  used  should  be  of  a  good  quality,  and, 
if  it  is  desired  to  compare  results  with  those  ob- 
tained by  European  investigators,  it  is  well  to  use 
those  dyes  prepared  especially  for  Bacteria  investi- 
gation.1 A  small  piece  of  camphor  placed  in  the 
aqueous  solutions  of  aniline  dyes  does  not  impair 
their  staining  qualities,  and  tends  to  prevent  the 
growth  of  moulds.  These  solutions  must,  how- 
ever, always  be  filtered  before  using. 

Cover-glass  Preparations,  for  the  study  of  Bac- 
teria contained  in  fluids,  by  fixing,  hardening,  and 

1  A  collection  of  the  more  important  aniline  dyes  can  be  ob- 
tained of  Dr.  Georg  Griibler,  ' '  Physiologisch-Chemisches 
Laboratorum,"  Leipsic,  Durfour  Strasse,  No.  17,  for  about 
$4.00,  by  ordering  through  any  of  the  New  York,  Philadelphia, 
or  Boston  drug  houses,  or  by  sending  a  money-order  direct  to 
Dr.  Griibler. 


40  THE  TECHNOLOGY  OF 

staining  them.  Always  make  more  than  one 
preparation,  usually  three.  Place  a  small  quantity 
of  the  fluid  to  be  examined  upon  a  sterilized  cover- 
glass  (i.  e.,  one  which  has  been  passed  two  or 
three  times  through  a  spirit  flame),  cover  it  with 
another  and  slide  the  two  apart  (do  not  lift  them 
apart).  Then  place  them  in  a  drying-oven,  at  a 
temperature  of  120°  C.  Simple  exposure  to  the 
air  will  answer  in  some  cases.  By  far  the  best 
method,  however,  since  the  Bacteria  and  cellular 
elements  contained  in  a  thin  layer  of  fluid  are  not 
injured  by  rapid  drying,  is  to  seize  the  cover-glass 
upon  which  is  spread  the  fluid  to  be  examined,  with 
the  pincers,  and  to  pass  it  three  or  four  times  with 
the  prepared  side  up,  to  and  fro  through  the 
flame  of  a  Bunsen  burner  or  of  a  spirit-lamp, 
"  about  as  fast  as  you  would  cut  bread "  (Rind- 
fleish).  After  a  few  trials  one  learns  how  to  do 
this  without  burning  the  preparation.  This  heat- 
ing coagulates  any  albumen  present,  and  prevents 
the  specimen  swelling  up  in  the  subsequent  treat- 
ment. When  it  is  desired  to  retain  all  the  finer 
details  of  structure,  which  are  usually  lost  by 
drying,  specimens  may  be  fixed  and  hardened  by 
exposing  them  to  the  fumes  of  osmic  acid,  or,  still 
better,  to  the  fumes  of  absolute  alcohol.  Pour  a 
few  drops  of  absolute  alcohol  into  a  deep  watch- 
glass  ;  suspend  the  cover-glass  over  this,  with  the 
prepared  side  up ;  place  a  second  watch-glass  over 


BACTERIA    INVESTIGATION.  41 

the  whole,  and  take  it  all  up  with  the  pincers  and 
warm  it  over  the  flame  until  it  steams  slightly. 
Besides  the  above,  the  ordinary  methods  of  hard- 
ening may  be  employed,  —  chromic  or  picric 
acids,  or  their  combinations  with  other  acids.  The 
prepared  cover-glasses  should  be  thoroughly  rinsed 
of  these  acids  before  staining. 

General  Methods  for  Staining  Cover-Glass  Pre- 
parations. —  In  those  cases  when  it  is  not  desired 
to  show  the  characteristic  manner  in  which  certain 
pathogenic  Bacteria  behave  towards  staining  fluids, 
the  preparations  may  be  stained  with  Bcehmer's 
hsematoxylin  [F.  22.]  with  methyl-blue  or  with  a 
concentrated  aqueous  solution  of  vesuvin  [F.  38.]. 
Where  a  more  intense  color  is  desired,  or  in  work- 
ing with  those  forms  which  do  not  stain  readily 
methyl-blue  [F.  23.],  gentian-methyl-violet  [F. 
18.],  or  acid  fuchsin  [F.  37.]  may  be  used  in 
connection  with  a  watery  solution  of  aniline  oil, 
[F.  5.].  In  any  of  the  above  (Koch's  Method) 
allow  the  cover-glass  to  float  prepared  side  down 
upon  the  surface  of  a  quantity  of  the  staining  fluid 
in  a  watch-glass,  for  from  two  minutes  to  twenty- 
four  hours  (raising  the  temperature  of  the  staining 
fluid  shortens  the  time  required) ;  remove,  and  wash 
in  acidulated  water  [F.  7.]  until  the  color  has 
apparently  faded  out,  pass  rapidly  through  absolute 
alcohol,  dry,  and  mount  in  Canada  balsam  (free  from 
chloroform)  or  acetate  of  potash  solution  [F.  34.]. 


42  THE   TECHNOLOGY  OF 

Of  all  general  methods  for  staining  Bacteria  in 
cover-glass  preparations,  Friedlander  considers 
that  of  Dr.  Gram,  of  Copenhagen,  to  he  hy  far 
the  best  and  nearest  perfection.  It  is  especially 
useful  where  the  material  to  be  investigated  is 
rich  in  nuclei ;  as  blood,  pus,  and  the  splenic  or 
hepatic  juices.  It  allows  of  an  isolated  staining 
of  the  Bacteria,  everything  else  in  the  field  re- 
maining colorless  ;  the  Bacteria  being  so  intensely 
colored  a  deep  dark  blue  that  the  investigator  must 
find  every  single  individual  present. 

Gram's  Method.  —  The  prepared  cover-glasses 
are  allowed  to  float  prepared  side  down  upon 
a  quantity  of  filtered  Balmer-Franzel's  aniline- 
gentian-violet  solution  [F.  19  a.]  which  is  in  the 
meantime  held  over  a  flame  and  warmed.  (The 
preparations  used  in  this  method  must  have  pre- 
viously been  in  absolute  alcohol,  not  in  water  or 
dilute  alcohol.)  They  are  then  placed  for  one  min- 
ute in  iodo-iodide  of  potash  solution  [F.  35  d.], 
then  washed  in  absolute  alcohol  (1-3  min.)  until 
the  color  becomes  invisible  to  the  naked  eye, 
clear  up  in  oil  of  cloves,  and  mount  in  xylol- 
Canada  balsam  or  in  glycerine-jelly  [F.  20.]. 
Special  methods  for  staining  "  cover-glass "  pre- 
parations will  be  found  in  their  appropriate  place 
under  the  treatment  of  pathogenic  Bacteria.  Beau- 
tiful double-stainings  may  be  produced  by  the 
use  of  Bismarck-brown  after  Gram's  method,  i.  e.9 


BACTERIA    INVESTIGATION.  43 

after  being  stained  violet  and  washed  in  the  potash 
solution  and  alcohol,  place  the  cover-glasses  for  a 
moment  in  Bismarck-brown  or  vesuvin  solution, 
then  wash  in  absolute  alcohol,  pass  through  oil  of 
cloves,  and  mount.  The  Bacteria  remain  a  dark 
blue,  while  the  nuclei  become  a  yellowish  brown. 
The  Preparation  of  Bacteria  in  and  upon  Firm 
/Substances.  —  If  the  tissue  to  be  examined  is  a 
fresh  one,  cut  sections  of  it  with  a  freezing  micro- 
tome or  a  Valentin's  knife,  and  pass  them,  before 
staining,  through  absolute  alcohol.  Hardened 
tissues  may  be  cut  in  serial  sections  by  the 
microtome,  and  left  in  alcohol  until  desired  for 
staining.  Only  small  pieces  of  tissue  should 
be  taken  for  hardening,  and  should  be  placed  in 
relatively  large  quantities  of  alcohol.  Hardening 
in  chromic  acid  or  Miiller's  fluid  is  not  well  suited 
for  the  study  of  Bacteria,  since  these  fluids  are 
liable  to  produce  in  the  tissues  numerous  dark 
granules  which  are  difficult  to  clear  up.  The 
hardening  must  not  be  continued  too  long,  as 
many  Bacteria  lose  their  capacity  for  taking  stain 
after  being  three  or  four  days  in  alcohol.  Neither 
must  the  specimens  to  be  cut,  be  imbedded  in 
paraffine,  as  this  and  the  volatile  oil  requisite  to  the 
imbedding  process  destroys  the  staining  capacity  of 
the  microbes.  Transparent  soap  [F.  41.]  or  cel- 
loidin  [F.  42.]  may  be  used  as  imbedding  masses. 
Two  general  plans  are  open  for  staining  the  Bac- 


44  THE  TECHNOLOGY  OF 

teria  which  exist  in  sections  of  firm  substances, 
i.  e.,  that  in  which  but  one  color  is  used,  and  the 
tissue  substance  afterwards  bleached  out,  leaving 
the  stained  Bacteria  upon  a  transparent  ground, 
or  that  in  which  a  double  staining  is  employed, 
the  microbes  retaining  one  color  and  the  tissue 
another.  After  the  first  plan  we  have  — 

Weigert's  Method,  according  to  which  a  solution 
of  any  basic  aniline  dye  may  be  used,  methyl- 
blue  [F.  23.],  methyl-violet  [F.  25.],  gentian- 
violet  [F.  18.],  rosanilin-chlorhydrate  [F.  36.], 
magenta-red  [F.  26.],  fuchsin  [F.  16.],  Bis- 
marck-brown [F.  2.],  vesuvin  [F.  38.]  and 
aniline-brown  [F.  3.]  being  the  better,  while 
iodine-green,  malachit-green  [F.  24.] ,  and  chrysoi- 
din  [F.  11.],  aniline-black  [F.  27.],  and  aniline- 
blue  are  less  preferred.  Allow  the  sections  to 
remain  for  18  to  24  hours  in  a  staining  fluid  pre- 
pared from  one  of  the  above ;  the  process  may  be 
hastened  by  warming  the  solution  at  45°  C. ;  it  is, 
however,  easy  to  over-stain  by  doing  this,  although 
such  an  accident  can  be  rectified  by  subsequent 
treatment  with  a  saturated  solution  of  carbonate  of 
potash.  After  staining,  wash  in  distilled  water, 
then  place  for  5  minutes  in  50  per  cent  alcohol, 
and  then  for  15  minutes  in  absolute  alcohol,  in 
order  that  all  water  may  be  removed,  and  the 
tissue  decolorized.  Finally  clear  up  in  oil  of 
cloves,  oil  of  bergamot,  or  xylol,  and  mount  in 


BACTERIA    INVESTlGATIONr  45 


xylol-Canada  balsam.  Another  plan  adapted  to 
all  forms  of  Bacteria  is  — 

Schutz'  Method,  which  can  be  used  in  sections 
of  fresh  tissues  cut  with  the  freezing  microtome. 
Place  the  sections  for  twenty-four  hours  in  a 
methyl-blue  solution  [F.  23.],  wash  in  water  to 
which  a  few  drops  of  acetic  acid  have  been  added, 
then  in  50  per  cent  alcohol  for  5  minutes,  absolute 
alcohol  15  minutes,  clear  up  in  cedar  oil,  mount  in 
Canada  balsam. 

Babes  recommends  the  use  of  safranin  for  stain- 
ing sections  of  pathological  specimens  containing 
Bacteria.  The  sections,  hardened  in  alcohol  or 
chromic  acid,  should  be  steeped  in  a  super-satu- 
rated solution  of  saffranin,  which  is  warmed  to 
60°  C.  and  filtered  in  this  state.  The  sections  are 
placed  in  a  small  quantity  of  the  liquid  in  a  watch- 
glass,  which  is  then  warmed  for  a  few  seconds  over 
ji  spirit  lamp  until  the  precipitating  crystals  are 
re-dissolved  ;  the  sections  are  left  for  a  minute, 
then  washed  in  water,  and  dehydrated  in  absolute 
:ilcohol  as  quickly  as  possible,  then  transferred 
to  turpentine,  and  mounted  in  balsam.  Every 
Micrococcus  appears  brownish-red,  while  the  sur- 
rounding tissue  assumes  a  fine  rose-red  ;  the 
jSacilli  of  tuberculosis  and  lepra  are  not  thus 
gained. 

According  to  the  second  general  plan,  that 
of  Double  Staining,  we  may,  after  removing  from 


46  THE   TECHNOLOGY   OF 

the  tissue  the  color  first  employed,  pass  the  sec- 
tions through  a  solution  of  some  other  aniline 
dye :  the  browns  are  to  be  preferred,  e.g.,  Koch's 
"  glycerine-aniline-brown "  [F.  3.].  It  is  not  abso- 
lutely necessary  to  remove  the  first  color  from  the 
substance  of  the  tissue,  as  this  will  retain  the  color 
to  which  it  is  last  subjected ;  the  sections  may 
therefore  be  passed  at  once  from  the  first  staining 
fluid  into  a  second  filtered  aqueous  solution  of 
almost  any  of  the  basic  aniline  dyes,  which  will 
change  the  color  of  the  tissue,  while  the  Bacteria 
retain  the  color  to  wrhich  they  were  first  subjected. 
Of  course  only  complementary  colors  should 
be  chosen  for  double  staining.  The  following 
make  good  combinations,  methyl-blue  and  vesu- 
vin  [F.  23,  38.]  (used  by  Koch  in  cover-glass 
preparations),  gentian-violet  and  vesuvin  [F. 
38.],  or  picrocarmine-fuchsin  and  methyl-blue 
[F.  23.]  or  Subbotiria  Combination,  in  which  he 
took  preparations  wrhich  had  been  hardened  by 
means  of  osmic  acid  fumes  [F.  30]  or  with 
chromic  acid,  and,  after  washing,  treated  them  with 
a  one  per  cent  aqueous  solution  of  methyl-green 
[F.  24.]  for  about  two  hours,  and  subsequently 
with  picro-carminate  of  ammonia  [F.  33.],  washed 
them  again,  dried  and  mounted  them  in  Canada 
balsam.  The  Bacteria  appeared  green,  while  th§ 
tissue  was  stained  red. 

Mounting.  —  Such  specimens  as  are  desired  for 


BACTERIA    INVESTIGATION.  47 

purposes  of  photo-micrography,  if  colored  in  vesu- 
vin  (best  adapted) ,  should  be  mounted  in  glycerine, 
or  glycerine-jelly  may  be  used  [F.  20.],  which  be- 
comes fluid  by  slight  warming,  and  stiffens  again 
when  cool ;  if  not  colored  with  vesuvin,  the  satu- 
rated solution  of  acetate  of  potash  [F.  34.] 
should  be  used  for  mounting  specimens  to  be 
photographed..  The  cover-glasses  can  be  immedi- 
ately secured  in  place  in  glycerine  mounts  by 
touching  a  heated  wire  to  a  piece  of  paraffine  or 
wax,  some  of  which  will  form  a  drop  at  the  end 
of  the  hot  wire  ;  allow  this  to  fall  at  one  corner 
of  the  cover-glass,  thus  you  fix  the  latter  so  that 
it  is  not  readily  moved  while  more  paraffine  or 
wax  is  applied,  and  spread  over  the  edges  with 
the  hot  wire,  or,  better  than  paraffine  or  wax  is 
the  cement  recommended  by  Prof.  Csokor  [F. 
12.].  These  are  all  preferable  to  "gold  size," 
since  by  their  use  the  cover-glass  can  be  washed 
and  wiped  in  a  few  minutes  after  mounting  with- 
out fear  of  displacing  it.  In  place  of  these  sub- 
stances a  good  thick  solution  of  shellac  in  alcohol, 
or  of  Canada  balsam  in  chloroform,  or  asphalt 
varnish  may  be  used.  Aniline  colors,  other  than 
vesuvin,  do  not  keep  well  when  mounted  in 
glycerine ;  Canada  balsam  is  therefore  used  in 
most  preparations,  which,  if  they  have  been  treated 
with  aqueous  staining  fluids,  must  be  dehydrated 
by  drying,  or  by  passing  through  absolute  alcohol, 


48  THE  TECHNOLOGY   OF 

then  cleared  up  with  oil  of  cloves,  oil  of  bergamot, 
or  xylol  [F.  29.],  and  finally  mounted. 

PREPARATION  OF  BACTERIA  FOR  PHOTOGRAPHING. 

Sternberg's  Method  of  preparing  Bacteria  for 
photographing.  The  Bacteria  are  dried  upon  a 
slide  or  a  thin  glass  cover,  and  are  then  treated 
with  commercial  sulphuric  acid,  a  drop  of  which 
is  placed  upon  them.  After  two  or  three  minutes 
the  acid  is  washed  off  by  a  gentle  stream  of  water, 
and  the  Bacteria  are  then  covered  with  an  aqueous 
solution  of  iodine  (iodine,  grs.  3,  potassic  iodide, 
grs.  5,  water,  grs.  500).  After  a  few  minutes  they 
will  be  found  to  present  a  deep  orange  or  brown 
color,  which  gives  the  desired  contrast  in  a  photo- 
graph negative.  This  method  is  only  useful  for 
extemporaneous  preparations  which  are  to  be  pho- 
tographed immediately.  The  color  fades  after  a 
time,  and  the  Bacteria  undergo  changes  in  form 
(swelling)  as  a  result  of  this  treatment,  which 
renders  the  method  unsatisfactory  when  the  object 
is  to  make  a  permanent  preparation.  For  this 
purpose  nothing  is  better  than  aniline-violet,  which, 
indeed,  leaves  nothing  to  be  desired  when  a  col- 
lection is  being  made  without  reference  to  photo- 
graphy. The  specimens  should  be  mounted  either 
in  a  solution  of  acetate  of  potash  (Koch's  method), 
or  preferably  in  carbolic  acid. 

KaschkcCs    Method  of  preparing   Bacteria  for 


BACTERIA    INVESTIGATION.  49 

photographing.  After  the  drop  containing  the 
Bacteria  is  dried  upon  the  slide  in  the  usual  man- 
ner, the  spot  is  moistened  with  a  metallic  solution  of 
an  iodide  (cadmiumiodide  1  :  50  Vas  employed), 
and  in  two  or  three  minutes  the  Bacteria  are  suffi- 
ciently iodized.  The  slide  is  then  carefully  and 
rapidly  washed  in  distilled  water,  and  immediately 
flowed  with  a  few  drops  of  silver  solution  from  the 
negative  bath.  If  the  right  time  has  been  hit,  and 
the  iodide  has  not  acted  for  too  short  a  period, 
and  the  washing  has  not  been  continued  too  long, 
the  contour  of  the  dried  drop  will  be  seen  to  show 
a  slight  yellow  color,  due  to  the  iodide  of  silver 
which  is  formed.  Only  an  exceptionally  short 
exposure  to  light  is  sufficient,  after  which  the 
developer  (strongly  acidified  and  dilute  iron  de- 
veloper) is  added,  and  the  drop  suddenly  becomes 
black.  After  thorough  washing,  the  deeply-colored 
Bacteria  are  mounted  in  balsam,  and  may  then  be 
readily  photographed.  This  method  of  staining  is 
only  useful  for  photographic  purposes,  and  there 
is  some  chance  of  mistaking  fine  silver  precipitates 
for  Micrococci  or  other  forms.  In  case  of  any 
doubt  of  this  kind,  the  original  forms  should  be 
stained  with  aniline  colors  and  examined  in  the 
usual  way. 

Dufrenne's  Method  of  photographing  Bacteria 
stained  with  fuchsin.  M.  Dufrenne  describes  the 
process  which  he  adopts  for  photographing  Bac- 


50  THE  TECHNOLOGY   OF 

teria  with  a  Tolles  l-10th  inch  (homogeneous 
immersion)  objective,  without  eye-piece,  and  the 
use  of  extra  rapid  bro mo-gelatine  plates,  devel- 
oped with  ferro-oxalate ;  a  petroleum  lamp  being 
employed  for  illumination.  "If,"  he  says,  "the 
determination  of  the  actinic  focus  of  objectives 
constitutes,  so  to  say,  the  chief  difficulty  in  photo- 
graphing microscopic  preparations,  it  is  no  longer 
so  when  we  deal  with  organisms  so  infinitely  strong 
as  Bacilli.  Here  arises  a  difficulty  of  quite  another 
kind,  which  at  first  seemed  insurmountable  —  the 
staining  the  Bacilli  by  means  of  fuchsin.  This 
agent,  even  when  it  is  employed  in  thick  layers, 
is  somewhat  actinic,  and  it  becomes  more  so  as 
the  object  stained  is  smaller  or  more  transparent. 
These  two  circumstances  are  combined  in  the  high- 
est degree  in  the  organisms  in  question.  Thus,  at 
the  beginning,  the  plates  exposed  were  either  uni- 
formly acted  on,  or  the  image  was  so  faint  and  so 
little  differentiated  after  development,  that  they 
were  worthless  for  proofs  on  glass  or  on  paper. 
These  negative  results  suggested  the  abandonment 
of  the  attempt,  when  the  idea  was  suggested  of 
having  recourse  to  the  use  of  a  compensating  glass 
of  a  color  complementary  to  red  (that  is,  green), 
placed  between  the  objective  and  the  sensitized 
plate;  By  thus  filtering  the  image  formed  by  the 
objective,  the  red  rays  —  the  only  ones  passing 
through  the  Bacilli  —  are  absorbed,  if  not  wholly, 


BACTERIA    INVESTIGATION.  51 

at  least  in  great  part.  The  microbes,  therefore, 
appear  nearly  black  on  the  plate,  and  make  a 
much  slower  impression  than  the  rest  of  the  pre- 
paration which  gives  free  passage  to  all  the  green 
rays.  More  contrast  is  thus  obtained,  and  a  very 
distinct  photograph  produced." 

LITERATURE    ON   THE    PREPARATION   AND    STUDY  OF 
BACTERIA    IN    GENERAL. 

ABB£  :  "  Ueber  Blutkorperzahlung,"  Sitzungs- 
bericht  der  Jenaischen  Gesell.  f.  Med.  u.  Natur- 
wissenschaft.  1878.  No.  29. 

ADAMS  (J.  M.)  :  "Easy  Method  of  Staining 
Bacteria."  The  Microscope,  IV.  (1884),  pp.  224, 
225. 

BABES  (Y.)  :  "Ueber  einige  Farbungsmethoden, 
besonders  fur  krankhafte  Gewebe,  mittlst,  Sat  ra- 
ni n  imd  deren  Resultate."  Arch.  f.  mikr.  Anat. 
XXII.  (1883),  pp.  356-65. 

BIENSTOCK  :  (On  staining).  Zeitschrift.  f.  klin. 
Med.  1884,  p.  1. 

BLANCHARD:  (On  method  of  staining).  Rev. 
Inter.  Sci.,  III.,  1879,  p.  245.  — Journ.  Roy.  Mic. 
Soc.,  II.,  1879,  p.  463. 

BUCHNER  :  "  Ueber  das  Verhalten  der  Spaltpilz- 
Sporen  zu  den  Anilinfarben."  Aerztliches  Intelli- 
genzblatt.  1884.  No.  33,  p.  370. 

BURRILL  (T.  J.)  :  "  Preparing  and  Mounting 
Bacteria"  Proc.  Amer.  Soc.  Microscopists,  6th 


52  THE   TECHNOLOGY   OF 

meeting,  p.  79,  also  Micros.  News,  vol.  IV.,  1884, 
No.  44,  p.  199. 

DUFRENNE  :  (Improvement  in  photographing 
Bacteria).  Bull  Soc.  Belg.  Micr.  X.,  1884, 
pp.  128-32.  Journ.  Roy.  Mic.  Soc.,  1884,  p.  fi27. 

EHRLICH  (P.)  :  I.  "Beitrage  sur  Kenntnis  der 
Anilinfarbungen."  Arch.  f.  mikr.  Anat.,  Bd.  13. 
S.  263.  1876.  II.  "Ueber  das  Methylenblau 
und  seine  klinische  bakterioseopische  Verwen- 
dung."  Zeitschr.  f.  klin.  Med.  II.  p.  70,  1881. 

FLESCH  :  "  Beleuchtungsvorrichtungen  zum  Mi- 
kroskopiren  bei  kunstlichem  Lichte."  Sitzber.  d. 
phys.  med.  Gesellsch.  zu  Wiirzburg,  1882, 
p.  37. 

FOL  :  "Lehrbuch  der  mikroskopischen  Anato- 
mie  "  Erste  Lieferung.  Die  Mikroskopisch-anato- 
mische  Technik."  p.  202-208.  Leipzig  (Wm.  En- 
gelmann)  1884. 

FRIEDLANDER  (C.)  :  "Microscopische  Technik, 
zum  Gebrauch  bei  medicinischen  und  pathologisch 
anatomischen  Untersuchungen,"  2  Aufl.  Berlin, 
1884. 

GIBBES  (H.)  :  "Double  staining  for  Bacilli." 
The  Lancet,  1883,  p.  771. 

GRAM  (C.)  :  "  Ueber  die  isolirte  Farbung  der 
Schizomyceten  in  Schnitt-  und  Trochen-prapara- 
ten."  Fortschritte  der  Med.  Bd.  2,  No.  6, 
p.  185,  1884. 

HANSEN  (E.  C.)  :    "Ueber  das  Zahlen  mikros- 


BACTERIA    INVESTIGATION.  53 

kopischer  Gegenstande  in  der  Botanik,  "  6  wood- 
cuts. Zeitschr.  f.  wiss.  Mikroskopie  Bd.  I.  H. 
2,  p.  191. 

HAZLEWOOD  (F.  T.)  ;  (On  a  blue  staining  fluid 
for  Bacteria).  American  Monthly  Microscopical 
Journal,  1884,  p.  83  ;  also  June,  1883. 

HOFFMANN  (G.  v.)  :  "  Untersuchungen  iiber 
Spaltpilze  im  menschlichen  Blute"  82  pp.  8°. 
mit  2  Tafln.  Berlin.  (Hirschwald),  1884. 

HUEPPE  (F.)  :  "Die  Methoden  der  Bakterien- 
Forschung."  Mit  2  Tafln  in  Farbendruck  und 
31  Holzschnitten.  February,  1885.  Wiesbaden, 
(C.  W.  Kreidel's  Yerlog). 

KLEIN  (E.)  :  "  Micro-organisms  and  disease. 
An  introduction  into  the  study  of  specific  micro- 
organisms." London  (Macmillan),  1884,  8vo, 
108  woodcuts. 

KOCH  (R.)  :  "Zur  Untersuchung  von  pathoge- 
nen  Organismen."  Mittheilung  a.  d.  kais.  Gesund- 
heitsamt,  1881.  Bd.  1,  p.  1.  Cf.  Bed.  klin. 
Wochenschr.,  1882,  p.  15. 

KOCH  (R.)  :  "  Verfahren  zur  Untersuch.  z. 
Conserviren  und  Photographiren  d.  Bacterien." 
Beitrage  z.  Biol.  Bd.  II. 

LEE  (A.  B.) :  "  The  Microtomist's  Yade  Mecum. 
A  Handbook  of  the  Methods  of  Microscopic  Anat- 
omy." London  (J.  and  A.  Churchill),  1885, 
(Chapt.  XV.,  "Bacteria  Staining"). 

MARPMANN   (C.)  :    Die    Spaltpilze.   Grundziige 


64  .  THE  TECHNOLOGY  OF 

der  Spaltpilze  oder  Bacterienkunde "  (vid.  parts 
II.,  III.)  Halle  a.  S.  (Waisenhauser)  1884. 

MITTENZWEIG  :  "  Ueber  den  Vfcrlauf  des  bak- 
teriologischen  Cursus  im  Reichs  Gesundheitsamt " 
Conferenz,  d.  med.  Beamten  des  reg.  Bez.  Dus- 
seldorf.  Extr.,  in  Deutsch.  med.  Wochenschr. 
Nov.  20,  1884,  p.  769. 

NOWAK  (J.)  :  "Die  Infectionskrankheiten  vom 
aetiologisehen  und  hygienischen  Standpunkt.  Me- 
thoden  der  Untersuchung  auf  Microorganismen." 
Wien,  1884,  pp.  142. 

ORTH  (J.) :  "Compendium  der  pathologisch- 
anatomischen  Diagnostik,  nebst  Anleitung  zur 
Ausfulirung  von  obductionen  von  pathologisch- 
histologischer  Untersuchungen."  Dritte  neu  be- 
arbeite  und  mit  mikroskopische  Technicks  ver- 
mehrte  Auflage.  gr.  8,  1884. 

ORTH:  "Notizen  zur  Farbetechnik."  Berliner 
klin.  Wochenschr,  July,  1883,  p.  421. 

PARIETTI  (E.)  :  "  Recerche  relative  alia  pre- 
parazione  e  conservazione  di  Bacteri  e  d'Infusori  " 
Bollett.  Scientitico,  vol.  V.,  1883,  p.  95. 

PERTY  :  "  Zur  Kenntniss  kleinster  Lebensfor- 
men,"  1852,  p.  13. 

PEYER  (A.)  :  "  Die  Microscopic  am  Kranken- 
bette."  8vo.  Basel,  1884,  XII. ,  19  pp.  and  79  pi. 

PLAUT  (H.)  :  "Farbungs  Methoden  zum  Nach- 
weis  der  faulniss  und  pathogenen  Mikroorganis- 
nien,"  2  Aun.  Leipzig,  1885. 


BACTERIA    INVESTIGATION.  55 

RECKLINGHAUSEN  :  (On  rendering  Bacteria  more 
visible  by  acids  or  alkalies).  Verhandl.  der  physi- 
kal-medicin.  Gesellsch  in  Wurtzburg.  N.  F.  II. 
Bd.  Hft.  4,  1872.  Sitzungsbericht,  p.  XII. 

SAHLI  (H.)  :  "Ueber  die  Anwendung  von  Bo- 
raxmethylenblau  fur  die  Untersuchung  des  centra- 
len  Nervensystems  und  fiir  den  Nachweis  von 
Mikroorganismen,  speciell  zur  bacteriologischen 
Untersuchung  der  nervosen  Centralogane."  Zei- 
tschr.  f.  wiss.  Mikroskopie,  Bd.  II.  Hft.  1, 
p.  49. 

SlEDAMGROTZKY     U.    HOFMEISTER  :     "  Allleitung 

zur  mikroskopischen  und.  chem.  Diagnostik." 

STERNBERG  (G.  M.) :  "  Photo-Micrographs,  and 
How  to  Make  Them."  Boston  (J.  E.  Osgood  & 
Co.),  1883.  8vo,  pp.  204. 

STOWELL  (C.  H.) :  "  Bacillus  Staining."  The 
Microscope,  IV.,  p.  79,  1884. 

WALMSLEY  &  Co. :  "  Circular  on  Bacillus 
Staining."  The  Microscope,  vol.  III.,  p.  310 
(1883) ;  vol.  IV.  (1884),  pp.  79,  80. 

WEIGERT  :  "  Zur  Technik  d.  mikrosk.  Bakterien 
Untersuch."  Virchow's  Archiv.  Bd.  84,  p.  283, 
1881.  Cf.  Sitzung  der  Schlesischen  Gesellschaft 
fiir  vaterlandische  Cultur  vom  10  Dec.,  1875. 
Berl.  klin.  Wochenschr,  1877.  No.  18  u.  19.- 
Bericht  iiber  die  Munchener  Naturforschersver- 
sammlung,  1877,  p.  283. 

ZACH ARIAS    (O.)  :    "Das  Mikroskopie  und  die 


56  THE  TECHNOLOGY  OF 

wissenschaftlichen   Methoden   das   Untersuchung- 
en."     Leipzig,  1884. 

ZOPF  (W.)  :  "  Die  Spaltpilze  "  (Abschnitt  III. 
Methoden  der  Untersuchung) ,  2d  Edit.  Breslau. 
(Trewendt),  1884. 


II.  CULTURE  EXPERIMENTS. 

For  the  perfecting  of  this  method  of  investigat- 
ing Bacteria  we  are  chiefly  indebted  to  Dr.  Robert 
Koch.  In  it  we  have  a  plan  by  which  we  can  se- 
lect from  the  numerous  Bacteria  found  in  a  diseased 
body,  or  in  a  putrefying  or  fermenting  mass,  those 
forms  which  are  the  cause  of  the  change.  As  we 
have  already  noticed,  the  mucous  surfaces  of  the 
body  are  always  in  healthy  animals  the  home  of 
numerous  Bacteria  :  in  a  diseased  animal,  how  are 
we  to  find  the  potent  factor  of  the  morbid  condi- 
tion, how  isolate  it  from  the  forms  which  are  com- 
paratively innoxious  ?  Or,  if  our  attention  is  called 
to  some  milk  which  has  turned  blue,  as  it  some- 
times does  through  the  agency  of  Bacteria,  how 
are  we  to  select  from  the  myriad  of  living  forms, 
filaments,  rods,  etc. ,  —  in  short,  all  the  forms  which 
may  have  entered  the  milk  from  the  air  of  the 
stable  or  dairy,  — the  one  which  has  done  the  mis- 
chief? That  this  can  be  done  seems  scarcely  pos- 
sible ;  and  yet  it  is  not  so  difficult,  providing 
proper  care  is  exercised  in  the  processes  employed. 


BACTERIA    INVESTIGATION.  57 

For  instance,  in  the  case  of  the  blue  milk,  we  take 
a  small  quantity  of  the  milk  (what  will  stick  to  the 
point  of  a  cambric  needle)  ;  this  we  mix  with  about 
a  teaspoonful  of  properly  prepared  gelatine,  which 
becomes  fluid  at  35°  C.  In  this  melted  gelatine 
the  few  microbes  which  we  have  taken  up  on  the 
needle  become  scattered,  and  it  is  poured  out  upon 
a  flat  surface,  properly  protected  from  the  air,  and 
in  from  twenty-four  to  seventy-two  hours  we  obtain 
from  each  microbe  a  separate  pure  culture,1 — here 
a  mould-like  patch,  here  a  porcelain-like  fleck,  here 
a  red,  there  a  gray  or  blue,  —  all  pure  cultures. 
We  may  now  take  from  each  of  these  a  particle, 
as  we  did  from  the  milk,  and  make  a  new  sowing 
in  fresh  gelatine,  and  .by  acting  upon  some  pure, 
fresh  milk  with  the  various  pure  cultures,  we  find 
at  last  that  those  Bacteria  belonging  to  the  little 
bluish  patch  are  the  special  agents  in  the  case. 
After  this  plan  of  procedure,  — but  not,  of  course, 
in  so  simple  a  way,  —  did  Koch  work  out  the 
"  Comma  Bacillus "  of  cholera,  the  Bacillus  of 
septicaemia  in  mice,  and  others,  the  specific  germs 
of  pyaemia,  erysipelas,  pneumonia,  etc.  "  It  is 
only  by  such  monosporus  cultivations,"  writes 
Prof.  Lankester,  "  that  we  can  arrive  at  solid 
conclusions  in  reference  to  the  forms  and  activi- 
ties of  Bacteria,  e.g.,  as  to  whether  one  form  can 
give,  rise  to  progeny  of  another  form,  when  its 
food  and  conditions  of  growth  are  changed,  and 


58  THE  TECHNOLOGY   OF 

again  as  to  whether  fermentative  powers  can  be 
lost  or  acquired  in  the  course  of  generations 
derived  from  one  parent  germ,  but  subjected  to 
different  conditions  as  to  food,  temperature,  and 
oxygen.  The  method  of  gelatine  cultivation  de- 
vised by  Dr.  Koch  places  the  means  of  following 
out  these  inquiries  in  the  hands  of  every  skilful 
microscopist." 

Again,  Bienstock,  in  connection  with  the  state- 
ment that  it  is  impossible  to  establish  the  identity 
of  two  micro-organisms  simply  from  the  resem- 
blance of  their  morphological  characters,  says : 
"In  bacterial  observations,  culture,  and  not  the 
microscope,  is  the  important  point.  The  mi- 
croscope is  principally  only  an  accessory  check- 
ing apparatus.  It  gives  exact  information  only 
in  the  study  of  morphology ;  it  is  an  uncertain 
guide,  and,  in  physiology,  generally  of  no  use 
at  all." 

Pasteur  expresses  himself  regarding  culture 
methods  as  follows :  "  Une  methode  pour  ainsi 
dire  unique  m'a  servi  guide  dans  1'etude  des 
organismes  microscopiques.  Eile  consiste  essen- 
tiellement  dans  la  culture  de  ces  petits  etre  a 
1'etat  de  purete,  c'est-a-dire,  degages  de  toutes 
les  matieres  heterogenes  mortes  ou  vivantes  qui 
les  accompagnent.  Par  1  'emploi  de  cette  methode, 
les  questions  les  plus  ardues  regoivent  parfois  des 
solutions  faciles  et  decisives."  Although  many 


BACTERIA    INVESTIGATION.  59 

microbes  seem  to  flourish  in  solutions  of  inorganic 
salts,  it  is  found  preferable  to  have  the  medium  in 
which  the  cultures  are  carried  on  rich  in  organic 
materials,  containing  at  the  same  time  some  inor- 
ganic salts.  Successful  cultures  depend  upon  the 
use  of  a  completely  sterilized  nourishing  medium 
and  a  pure  sowing.  These  culture  media  may  be 
either  of  a  solid  or  liquid  character,  each  having  its 
own  particular  advantage  and  use.  No  medium 
can  be  prepared,  however,  which  will  germinate 
all  forms  of  Schizomycetes  indiscriminately;  e.g., 
Micrococcus  urece  will  not  germinate  in  broths,  but 
in  urine,  while  the  microbes  of  chicken  cholera 
will  die  within  forty-eight  hours,  if  placed  in  (1  'eau 
de  levure)  a  decoction  of  beer  yeast,  which  forms 
an  excellent  culture  fluid  for  other  forms,  espe- 
cially for  filth  Bacteria. 

Fluid  Culture  Media  [F.  1.13.31]. —Broths 
made  from  all  kinds  of  flesh,  from  that  of  human 
beings  to  that  of  horses,  have  been  used  by  various 
investigators ;  these  are  freed  from  fat,  neutral- 
ized, filtered,  and  sterilized.  Decoctions  and  infu- 
sions of  various  vegetal  substances  are  used  under 
special  circumstances.  Sterilizing  the  culture  me- 
dium is  accomplished  ordinarily  by  the  use  of  heat 
sufficient  to  kill  all  germs  or  fully  developed  Bac- 
teria in  the  fluid.  It  may  be  accomplished  in 
bulk,  and  then  transferred  to  sterilized  culture 
vessels,  or  it  may  be  placed  in  these,  hermetically 


60  THE  TECHNOLOGY  OF 

sealed,  or  simply  stopped  with  a  plug  of  cotton, 
and  then  sterilized. 

For  sterilizing  the  culture  fluid  in  bulk,  a  strong 
iron  vessel  is  used,  having  (like  a  Wolffs  bottle) 
two  openings  through  the  close-fitting  top.  One  of 
these  is  the  opening  of  a  tube,  closed  at  its  lower 
end,  for  holding  the  thermometer ;  in  the  second 
opening  a  metallic  tube  is  fitted  by  being  tightly 
wrapped  in  cotton  and  pressed  firmly  into  the 
opening.  One  end  of  this  tube  extends  into  the 
vessel,  while  the  other  is  bent  twice  around  and 
joined  to  a  bit  of  rubber  tubing,  which  is  held 
closed  by  a  wire  clamp.  To  sterilize  the  culture 
medium,  draw  up  the  bent  tube  until  its  lower  end 
is  above  the  culture  fluid,  and  then  heat  the  whole 
up  to  110°  C.  for  about  an  hour.  Now  remove  the 
clamp,  and  allow  the  steam  to  pass  for  about  ten 
minutes.  Then  clamp  it  again,  and  press  the  bent 
tube  into  the  fluid.  If  it  is  now  desired  to  fill 
previously  sterilized  culture  vessels,  it  may  be  done 
by  relaxing  the  clamp,  when  the  fluid  will  flow 
out  through  the  tube. 

Pasteur's  Sterilization  Method  is  to  place  the 
culture  medium  in  small,  pear-shaped  flasks,  or 
balloons,  blown  from  a  glass  tube,  which  are  then 
hermetically  sealed  by  fusing  the  tube.  The  ves- 
sels thus  prepared  are  sunk  in  a  bath  containing 
chloride  of  lime  or  nitrate  of  soda  by  means  of 
a  perforated  metal  plate,  and  are  retained  here 


BACTEKIA    INVESTIGATION.  61 

for  twelve  hours  at  a  temperature  of  110°  C.  or 
115°  C. 

Buchner's  Sterilization  Method  requires  a  kettle 
20  ctm.  in  diameter  and  45  ctm.  deep,  which  can 
be  closed  steam-tight.  In  the  bottom  of  this  place 
a  layer  of  water  5  to  8  ctm.  in  depth.  Then,  by 
means  of  perforated  diaphragms,  the  test  tubes 
containing  the  culture  fluid  are  arranged  in  two 
tiers,  one  above  the  other ;  they  are  closed  with 
cotton,  and  each  has  also  a  bit  of  cloth  tied  over 
the  top,  and  a  small  funnel  is  inverted  over  each  to 
prevent  any  drops  of  water  from  reaching  the 
cotton.  The  cover  of  the  vessel  is  screwed  on 
tight,  and  the  whole  apparatus  heated  for  an  hour 
and  a  quarter,  and  then  kept  for  one  hour  at 
110°  C. 

TyndaWs  Method  of  Discontinuous  Heating. 
Tyndall  claims  that  it  is  only  possible  to  destroy 
the  germs,  or  "resting  spores,"  of  Bacteria  by 
exposing  them  to  alternate  heat  and  cold,  allowing 
in  the  cool  intervals  successive  broods  to  develop, 
and  killing  these  full-grown  microbes  at  each  heat- 
ing. Koch  modified  this  method  by  placing  the  de- 
gree to  which  the  heating  should  be  carried  at  70°  C. 

Miquel's  Method  of  Sterilizing  without  Heat 
depends  upon  the  use  of  filters.  For  some  rod- 
like  or  long,  filamentous  forms,  paper  has  been 
found  to  answer ;  but  to  k£ep  back  the  minute 
spores,  cylinders  of  clay  or  plaster  of  Paris  have 


62  THE   TECHNOLOGY  OF 

been  found  best  adapted.  A  flask  is  used,  in  the 
side  of  which,  near  the  neck,  a  small  vent-tube  has 
been  blown.  The  neck  of  the  flask  is  also  some- 
what constricted  at  its  lower  end.  A  mixture  of 
plaster  of  Paris  and  asbestos  [For.  39]  is  poured 
into  the  neck  of  the  flask,  and  allowed  to  dry 
slowly.  The  flask  is  then  heated  until  the  con- 
tained air  has  all  been  expelled,  and  any  germs 
killed.  The  vent-tube  is  then  fused  together,  and 
the  culture  fluid  poured  upon  the  plaster  plug  in 
the  neck  of  the  flask,  through  which  it  is  gradually 
forced.  This  method  is  used  for  such  culture  fluids 
as  contain  albumen,  which  would  be  coagulated  by 
the  heat  required  to  kill  the  germs  in  all  the  other 
methods  except  Koch's  modification  of  Tyndall's 
method. 

Gautier's  Method  of  Sterilization  without  Heat. 
He  employs  a  long-necked  flask,  made  of  "  fay- 
ence  "  or  of  unglazed  porcelain,  and  having  a  coni- 
cally  projecting  bottom.  Through  this  conical 
bottom  the  fluid  is  filtered  from  without  into  the 
flask.  Fastened  into  the  neck  of  the  flask  by 
means  of  cement  (F.  44)  is  a  glass  tube  bent  at 
right  angles,  of  which  one  limb  reaches  to  the 
bottom  of  the  cone,  while  the  other  outer  limb 
tapers  conically  and  passes  into  a  corresponding 
conical  expansion  of  a  second  tube.  This  second 
tube  is  likewise  bent  at  right  angles,  and  that  end 
not  united  with  the  first  tube  passes  to  the  bottom 


BACTERIA    INVESTIGATION.  63 

of  a  glass  flask  having  a  narrow  neck.  The  side 
of  this  glass  flask  carries  a  funnel-shaped  appen- 
dage. The  conical  openings  are  stopped'  with  cot- 
ton, and  the  whole  apparatus  sterilized  in  separate 
pieces.  The  tube  from  the  porcelain  flask,  after 
sterilization  and  the  removal  of  the  cotton,  is  fitted 
into  the  conical  expansion  of  the  second  tube.  In 
the  funnel-shaped  appendage  of  the  glass  flask  is 
fitted  a  tube  which  is  filled  with  sterilized  asbestos. 
All  the  joints  are  made  tight  with  shellac.  By 
aspiration  upon  the  free  end  of  the  asbestos  tube, 
the  air  in  the  entire  apparatus  is  rarified ;  and  if 
the  conical  bottom  of  the  porcelain  flask  is  placed 
in  a  fluid,  the  fluid  will  be  aspirated,  free  of  germs, 
into  the  flask. 

Koch,  Gaff  Icy,  and  Laeffler's  Steam  Sterilizing 
Cylinder.  —  This  consists  of  a  tin  cylinder  of 
about  i  meter  high  and  from  20  to  25  cm.  in 
diameter,  having  a  copper  bottom,  and  being  pro- 
tected against  loss  of  heat  by  an  asbestos  covering. 
At  the  lower  third  of  the  interior  is  a  grate,  the 
space  beneath  which  is  filled  three-fourths  full  of 
water.  This  is  caused  to  boil  by  from  three  to 
five  gas  flames  beneath  the  cylinder.  The  cylin- 
der is  closed  with  a  tin  top  covered  with  asbestos. 
This  top  does  not  fit  hermetically,  and  allows 
of  the  steam  escaping.  Through  its  apex  passes 
a  thermometer.  Advantage,  —  cheapness,  and  im- 
possibility of  temperature  rising  above  100°  C. 


64  THE   TECHNOLOGY  OF 

A  wire  basket,  fitting  into  the  cylinder,  is  used 
for  holding  the  tubes,  etc.,  to  be  sterilized,  facili- 
tating then-  introduction  and  removal. 

Solid  or  Gelatine  Culture  Media  are  made  by 
adding  to  any  of  the  fluid  culture  media  sufficient 
(5  :  10  per  cent)  pure  gelatine,  (F.  46  e.)  or,  pre- 
ferably, Japanese  sea- weed  glue,  "Agar-Agar," 
(F.  46  d.)  to  give  a  mass  which  will  become  fluid 
at  a  low  temperature,  e.g,,  30°  C. 

Koch  refers  to  solid  culture  media  as  follows 
(Deutsche  med.  TTochenschr.  No.  45,  1884,  p. 
726)  :  "In  the  ordinary  method  of  Bacteria  cul- 
ture in  a  firm  medium,  the  single  germs  must  be 
placed  as  for  as  possible  from  each  other,  in  order 
that  they  may  develop  entirely  apart.  The  mate- 
rial containing  the  Bacteria  is  therefore  placed  in  a 
gelatine  nourishing  medium  which  has  been  made 
fluid,  and  in  this  they  are  distributed  as  much  as 
possible,  and  then  are  poured  out,  with  the  gela- 
tine upon  them,  upon  a  glass  plate ;  the  gelatine 
hardening  quickly,  the  separate  Bacteria  scattered 
through  it  are  fixed  apart  from  each  other,  and 
each  germ  can  go  on  developing  and  multiplying 
until  it  becomes  a  mass  of  pure  culture,  visible  to 
the  naked  eye,  without  being  disturbed  by  or  mixed 
with  other  kinds.  The  principle  of  the  method  is 
to  develop  entire  colonies  from  single  individuals. 
It  is  much  more  difficult  to  separate  a  number  of 
different  kinds  of  microbes  on  the  surface  of  potato 


BACTERIA    INVESTIGATION.  65 

than  by  the  gelatine  process.  In  most  cases  the 
separating  of  pathogenic  from  non-pathogenic  Bac- 
teria upon  potato  cannot  be  accomplishe'd  because 
the  Bacteria  of  filth  grow  so  much  more  luxuriantly 
on  potato  that  they  soon  overcome  all  others.  Po- 
tato is  therefore  used  for  a  nourishing  sub-strata 
for  pathogenic  Bacteria  only  when  these  have  been 
obtained  from  pure  cultures  and  it  is  desired  to 
know  whether  they  will  subsist  on  vegetable  diet 
or  not." 

The  advantage  of  gelatine  cultures  consists  in 
the  ease  with  which  different  kinds  of  Bacteria  can 
be  isolated  and  studied  throughout  their  various 
phases  of  development.  When  a  test-tube  is  used 
as  a  culture  vessel  for  gelatine  media,  the  mass 
should  be  allowed  to  cool  while  the  tube  is 
inclined  at  an  angle  of  45° ;  it  will  thus,  when 
stiffened,  present  a  much  larger  surface  for  in- 
oculation. Thus,  after  the  sowing  instrument 
has  been  drawn  over  the  firm  surface  from  bot- 
tom to  top,  there  will  be  a  number  of  colonies 
spring  up  along  the  line  of  sowing,  and  from  any 
one  of  these  material  may  be  taken  for  new  pure 
cultures. 

Gelatine  offers  a  good  medium  for  the  study  of 
the  development  of  Bacteria,  since  the  germs  may 
be  sown  in  it  while  fluid,  and  then  spread  in  very' 
thin  layers  on  glass  slides,  which  are  kept  under  a 
bell-glass,  and  can  be  studied  as  they  are  with  low 


66  THE   TECHNOLOGY   OF 

powers,  or  with  immersion  lenses  by  placing  a  thin 
cover-glass  over  the  colony  to  be  investigated. 
The  great  objection  to  gelatine  culture  media  is 
that  they  cannot  be  retained  firm  at  those  tempera- 
tures best  adapted  to  the  growth  of  Bacteria,  30°- 
37°  C.  A  substitute  which  fulfils  this  requirement 
is  Koctis  Serum  Culture  Medium.  (F.  46  c.)  Se- 
rum of  an  ox  or  sheep  is  rendered  as  pure  as  possi- 
ble, and  placed  in  test-tubes  closed  with  cotton  and 
rubber  cloth  so  that  they  are  water-tight.  These  are 
for  six  days  heated  daily  for  one  hour  at  58°  C.,  by 
which  process  the  serum  is  in  most  cases  completely 
sterilized.  It  is  then  warmed  at  65°  C.  until  it 
becomes  stiff  and  firm.  After  this  treatment  the 
serum  appears  as  an  amber  yellow,  completely 
transparent  or  only  weakly  opalescent,  firm,  gela- 
tinous mass,  and  after  several  days  in  the  breeding 
oven  must  show  no  signs  of  developing  bacteria 
colonies.  If  the  heat  employed  in  stiffening  the 
serum  is  over  75°  C.,  or  lasts  too  long,  the  serum 
becomes  opaque.  To  furnish  a  large  surface  for 
culture,  the  serum  is  allowed  to  stiffen  while  the 
test-tube  is  in  an  inclined  position.  For  such  cul- 
tures as  are  to  be  studied  under  the  microscope,  the 
serum  is  allowed  to  stiffen  in  a  flat  watch-glass  or 
in  a  hollowed-out  slide.  Upon  this  stiffened  blood- 
serum,  which  forms  a  firm  nourishing  medium  at 
the  temperature  of  the  breeding  oven,  the  material 
to  be  investigated  is  placed,  and  the  whole  kept  at 


BACTERIA    INVESTIGATION.  67 

37°  or  38°  C.  until  development  takes  place,  which 
in  the  case  of  the  tubercle  Bacillus  is  from  twelve 
to  fourteen  days.  Instead  of  blood-serum,  hydro- 
cele  fluid  (F.  46  b.)  may  be  used,  being  rendered 
solid  in  the  same  way  as  blood-serum  ;  or  in  place 
of  either  of  these,  Miquel's  lichen  jelly  (F.  46  a.), 
which  is  convenient,  as  it  can  be  rendered  fluid 
again,  if  necessary,  which  is  not  the  case  with 
blood-serum  or  hydrocele  fluid. 

Culture  Vessels.  —  Ordinarily  simple  test-tubes 
closed  with  asbestos-wool,  or  with  cotton  are  used, 
but  if  it  is  desirable  to  be  especially  particular, 
take  a  conical-shaped  flask  of  good  glass,  with  a 
flat  bottom,  also  a  piece  of  glass  tubing  of  a  diam- 
eter smaller  than  the  neck  of  the  flask  and  some- 
what longer.  This  tube  must  be  slightly  pointed 
at  the  lower  end.  Pack  the  tube  three  quarters 
full  of  asbestos,  and  upon  the  top  of  this  place  a 
pad  of  cotton ;  wrap  the  tube  in  some  cotton  bat- 
ting and  press  it  firmly  into  the  neck  of  the  flask. 
This  apparatus  may  now  be  heated  up  to  200°  C. 
until  thoroughly  sterilized. 

Salmon's  Culture  Tube.  —  The  culture  tube  of 
Dr.  D.  E.  Salmon  consists  of  a  test-tube-like  body 
or  reservoir,  of  rather  heavy  glass,  about  four  or 
five  inches  in  length ,  and  three  quarters  of  an  inch 
internal  diameter.  Over  the  top  of  this  reservoir 
a  second  hollow  piece  or  cap  is  fitted.  Its  internal 
surface  is  ground  to  fit  snugly  over  the  ground 


68  THE  TECHNOLOGY  OP 

external  surface  of  the  upper  end  of  the  reservoir, 
thus  forming  a  ground  joint  union.  This  cap, 
about  two  and  a  half  inches  long,  abruptly  con- 
tracts near  its  middle  into  a  narrow  tube  of  about 
three  eighths  of  an  inch  internal  diameter.  The 
third  piece,  or  ventilating  tube,  is  like  an  inverted 
U,  one  limb  being  about  three  inches  long,  and 
one  and  a  half  inches  longer  than  the  limb  which 

O 

fits,  by  means  of  a  ground  joint,  over  the  narrow 
tube  of  the  cap.  The  longer  free  limb  of  the  ven- 
tilating tube  lodges  a  plug  of  glass-wool  from  one 
and  one  half  to  two  inches  long.  The  limbs  of  the 
ventilating  tube  are  about  one  inch  apart.  The 
culture  fluid  is  introduced  by  removing  the  cap, 
which  brings  with  it  the  ventilating  tube,  and  it  is 
sterilized  in  the  tube.  The  liquid  is  inoculated  by 
removing  the  ventilating  tube  only.  To  prevent 
the  ground  joints  from  sticking  too  firmly,  a  little 
sublimated  vaseline  is  introduced  between  the  sur- 
faces of  the  joints.  The  pipette,  used  to  introduce 
a  drop  of  fluid  containing  Bacteria,  consists  of  an 
ordinary  glass  tube  about  one  quarter  of  an  inch  in 
diameter,  and  two  or  three  inches  long,  one  end  of 
which  is  drawn  out  into  a  very  fine,  almost  capil- 
lary tube,  which  must  be  long  enough  to  easily 
reach  the  bottom  of  the  reservoir  when  introduced 
through  the  narrow  tube  of  the  cap ;  a  plug  of 
glass-wool  occupies  the  other  end,  which  is  closed 
by  a  rubber  ball.  The  method  of  inoculating  the 


BACTERIA    INVESTIGATION.  69 

culture  liquid  is  briefly  as  follows  :  The  pipette 
is  first  thoroughly  sterilized  by  flaming  every  por- 
tion of  it,  from  the  tip  of  the  capillary  tube  to 
near  the  rubber  ball,  until  the  contained  air  is 
subjected  to  a  temperature  of  at  least  150°  C.  It 
is  usual  to  bring  it  to  a  dull  red  heat,  avoiding  the 
contingency  of  melting  the  capillary  tube.  It  is 
hung  with  the  rubber  bulb  up,  to  avoid  its  capil- 
lary portion  coming  in  contact  with  anything  while 
cooling.  When  sufficiently  cool,  the  capillary  por- 
tion is  again  drawn  once  or  twice  through  the 
flame,  to  destroy  any  particles  that  may  have 
become  attached  meanwhile.  The  ventilator  of 
the  culture  tube  containing  the  Bacteria  to  be  sown 
is  flamed  and  removed,  and  the  narrow  tube  of  the 
cap  flamed ;  the  rubber  bulb  slightly  compressed 
and  the  pipette  introduced,  a  few  drops  drawn  up, 
the  pipette  slowly  withdrawn,  the  cap  flamed  again, 
and  the  ventilator  replaced.  The  cap  of  the  fresh 
tube  is  now  flamed  before  and  after  removing  the 
ventilator,  the  pipette  introduced,  a  drop  allowed 
to  fall  into  the  culture  fluid,  the  pipette  removed, 
the  narrow  tube  of  the  cap  again  flamed,  and  the 
ventilator  replaced.  When  the  source  of  the  Bac- 
teria is  an  exudate  or  the  flow  of  the  animal  body, 
various  methods  are  in  use  ;  the  method  given 
above  may,  however,  be  employed  in  most  cases. 
The  reservoir  may  be  variously  modified,  —  a  flask- 
shaped  body  may  be  used  for  cultures  that  require 


70  THE  TECHNOLOGY  OF 

an  abundance  of  air, — but  the  test-tube  form  will 
serve  nearly  all  purposes.  It  enables  the  nature 
of  the  opacity  of  the  liquid  to  be  readily  deter- 
mined, while  the  earliest  traces  of  a  membrane  or 
deposit  are  more  easily  detected  than  with  a  broad 
body  and  a  flat  bottom.  The  culture-tube  recom- 
mends itself  as  a  simple,  very  neat  apparatus, 
readily  filled,  sterilized  and  inoculated.  It  dis- 
penses with  the  troublesome  and  dangerous  expedi- 
ent of  disturbing  cotton  plugs,  and  of  tying  down 
various  air-filtering  materials.  It  is  easily  cleaned, 
and  hence  may  be  used  over  and  over  again,  — 
the  original  cost  of  the  tube  being  in  this  way 
reduced  to  a  minimum  in  the  end.  It  does  not 
break  readily,  nor  are  there  any  sharp  or  jagged 
edges  to  be  feared  in  the  manipulation  of  dangerous 
cultures.  It  is  very  compact,  and  occupies  but 
little  space  in  a  thermostat.  Finally,  the  chances 
of  contamination  through  the  air,  during  the  pro- 
cess of  inoculation,  are  practically  of  no  account. 

Sternberg's  Culture-flasks  are  made  from  glass 
tubing,  very  expeditiously,  and  at  small  expense. 
They  are  simply  little  bulbs  blown  from  a  glass 
tube,  and  having  a  long  neck  which  tapers  gradu- 
ally to  a  capillary  point.  Each  flask  contains  a 
sufficient  amount  of  nutrient  fluid  and  oxygen  to 
ensure  a  vigorous  and  abundant  development  of 
any  aerobic  micro-organisms  introduced  as  seed. 
The  Bacilli  readily  form  spores  in  these  flasks. 


BACTERIA    INVESTIGATION.  71 

When  properly  sterilized,  the  enclosed  culture- 
medium  remains  closed  indefinitely,  and  the  little 
flasks,  ready  for  use  at  a  moment's  notice,  may  be 
packed  away  in  drawers  or  boxes  for  years,  if 
desired,  and  may  be  conveniently  transported  from 
place  to  place.  The  inoculation  of  one  flask  with 
micro-organisms  contained  in  another,  or  with  a 
drop  of  blood  from  the  veins  of  a  living  animal, 
etc.,  is  effected  expeditiously,  and  with  perfect 
security  from  contamination.  Small  amounts  of 
fluid  may  at  any  time  be  withdrawn  from  one  of 
these  flasks  for  microscopic  examination,  without 
the  slightest  danger  of  introducing  foreign  organ- 
isms and  thus  destroying  the  purity  of  the  culture. 
Finally  these  little  flasks  take  the  place  of  a 
syringe,  where  an  inoculation  experiment  is  to  be 
performed,  the  contents  being  forced  beneath  the 
skin  of  a  living  animal,  by  applying  gentle  heat  to 
the  ball,  thus  causing  the  enclosed  air  to  expand, 
and  forcing  the  fluid  contents  through  the  capillary 
neck  of  the  flask. 

To  introduce  the  Sterilized  Culture  Fluid  into  the 
Sterilized  Culture  Vessel.  For  this  purpose  a  long, 
sharp  canula  is  needed,  made  of  silver,  platinum, 
or  glass.  Fasten  this  into  the  rubber  tube  of  the 
vessel  containing  the  culture  fluid  described  above, 
having  previously  sterilized  the  canula  by  holding 
it  in  the  steam  escaping  from  the  vessel,  or  by  heat- 
ing it  in  the  alcohol  flame.  Remove  the  cotton 


72  THE  TECHNOLOGY   OF 

plug  from  the  culture  vessel  (in  the  List-described 
form)  and  push  the  canula  through  the  asbestos  pad 
into  the  vessel ;  now  relax  the  clamp  on  the  rubber 
tube,  and  the  culture  fluid  will  flow  over  into  the 
culture  vessel ;  after  withdrawing  the  canula,  re- 
place the  cotton. 

To  sow  the  Microbes  in  the  Culture  Medium.  — 
Only  such  instruments  are  to  be  used  as  have  been 
sterilized  by  heat.  Since  hot  instruments  may  kill 
the  Bacteria,  and,  if  allowed  to  cool  in  the  air,  may 
be  again  contaminated,  a  plan  has  been  recom- 
mended whereby  a  number  of  cool  sterilized  wires 
may  be  kept  on  hand  (Fol.).  Take  an  ordinary 
test-tube  and  plug  it  with  asbestos,  and  pass  through 
this  plug  a  number  of  small  pipettes,  each  of  which 
is  stopped  with  an  asbestos  plug,  through  which 
passes  in  each  a  piece  of  platinum  wire.  Now 
sterilize  the  whole  tube  in  an  oven.  As  required, 
one  of  these  cool  sterilized  pipettes  and  wires  may 
be  removed  and  used.  Thrust  one  of  these  wires 
into  the  tissue  the  microbes  of  which  it  is  desired 
to  study,  draw  it  out,  and  in  the  meantime  having 
thrust  one  of  the  sterilized  pipettes  through  the 
asbestos  pad  into  the  culture  vessel,  pass  the  wire 
through  it  into  the  culture  fluid,  gelatine,  or  blood 
serum,  as  the  case  may  be,  and  the  sowing  is  com- 
pleted. If  the  sowing  is  to  be  made  from  a  fluid 
instead  of  a  tissue,  take  one  of  the  sterilized  pi- 
pettes without  the  wire,  and,  placing  over  it  a  rub- 


BACTERIA    INVESTIGATION.  73 

ber  cap,  draw  up  some  of  the  fluid  and  pass  through 
the  asbestos  pad  as  before.  Then  replace  the  cot- 
ton above  the  asbestos. 

To  carry  on  the  culture,  it  is  best,  with  fluid  media, 
to  keep  them  at  a  temperature  of  35°  C.  in  a  breed- 
ing oven.  This  is  also  the  plan  for  serum  cultures, 
but  gelatine  cultures  must  remain  in  the  room  at 
ordinary  temperatures,  at  which  only  do  they  re- 
main firm.  Otherwise  we  should  lose  the  advan- 
tages gained  by  the  addition  of  the  gelatine. 

Where  it  is  not  necessary  to  exercise  great  pre- 
cautions against  contamination,  as  in  growing  the 
various  pigment  Bacteria,  cultures  may  be  carried 
on  under  bell-glasses  by  sowing  the  Bacteria  upon 
slices  of  boiled  potato,  turnip,  on  eggs,  or  any 
similar  nourishing  substances,  as  the  gelatine  or 
Agar-Agar  masses  on  glass  slides  or  in  watch- 
glasses.  Place  a  piece  of  moistened  filter  paper 
upon  a  plate,  and  another  piece  in  the  dome  of  the 
bell-glass  ;  then  place  the  vegetable,  egg,  or  gela- 
tine with  its  Bacteria  on  the  plate,  cover  and 
keep  in  a  moderately  warm  place.  The  colonies, 
especially  in  the  case  of  pigment  Bacteria,  will 
soon  make  themselves  visible  to  the  naked  eye. 

Van  Tieghem  and  Lemonnier's*  and  MiqueTs 
Methods  for  the  cultivation  of  Bacteria  upon  the 
slide.  In  the  centre  of  an  ordinary  slide  is  fas- 
tened, by  means  of  Canada  balsam,  a  glass  ring 
from  4  to  5  mm.  thick,  cut  from  a  tube  used  for 


74  THE  TECHNOLOGY   OF 

organic  analysis,  and  the  cut  sides  properly  ground 
level.  A  thin  cover-glass,  round,  and  of  a  sufficient 
diameter  to  just  cover  the  ring  without  lapping  the 
edge,  is  fixed  on  the  upper  side  by  three  very 
small  drops  of  a  greasy  oil,  to  complete  the  cell. 
In  order  that  the  interior  air  may  be  always  satu- 
rated by  moisture,  a  few  drops  of  water  are  placed 
on  the  bottom  of  the  cell.  A  smalLdrop  of  nutri- 
tive liquid  is  suspended  at  the  centre  of  the  under 
surface  of  the  thin  cover.  In  this  drop  are  sown 
the  seed  microbes.  Such  was  the  moist  chamber 
used  by  Van  Tieghem  and  Lemonnier ;  it  has, 
however,  undergone,  during  the  last  ten  years, 
many  modifications,  more  or  less  satisfactory. 
Some  investigators  have  pierced  the  sides  of  the 
little  chamber  with  one  or  more  holes  for  facili- 
tating the  introduction  into  the  interior  of  various 
reagents.  For  the  study  of  the  atmospheric  Schi- 
zophytes,  Miquel  uses  the  same  cell  pierced  lat- 
erally by  an  opening  which  can  be  closed  by  a 
small  glass  rod  stopper.  The  cells  and  cover 
should  be  attached  to  the  slide  by  a  cement  that 
will  not  be  loosened  by  the  heat  used  to  sterilize 
the  chamber  [F.  44].  Then,  by  means  of  a 
pipette  with  a  curved  capillary  point,  the  steri- 
lized nutritive  liquid,  or  blood-serum,  broth,  urine, 
vegetable  juices,  etc.,  is  placed  upon  the  under 
surface  of  the  thin  glass  cover,  whilst  the  sowing 
of  the  organisms  whose  development  is  to  be 


BACTERIA    INVESTIGATION.  75 

watched  is  accomplished  by  the  aid  of  a  fine  plat- 
inum wire,  slightly  bent  at  the  point.  The  small 
rod  stopper  is  replaced,  and  the  whole,  with  the 
microscope,  is  placed  in  a  warm  chamber  at  30°  C. 
If  immersion  objectives  be  used,  a  little  glycerine 
can  be  added  to  the  water,  or  cedar  oil  used  on  the 
cover.  Good,  dry  objectives,  and  the  light  from 
a  paraffin  lamp,  will  generally  suffice  ;  but  Miquel 
gives  preference  to  the  excellent  No.  7  immersion 
objective  of  Nachet.  It  is  not  necessary  to  further 
describe  the  precautions  required  to  prevent  con- 
tamination, and  the  neglect  of  which  may  entirely 
nullify  the  value  of  the  cultivation.  Another  form 
is  one  in  which  there  are  two  opposite  lateral  open- 
ings into  the  cell ;  in  one  is  fitted  a  tube  contain- 
ing a  plug  of  cotton ;  through  the  other,  air  is 
projected  upon  the  drop  of  nutritive  fluid  on  the 
under  side  of  the  cover-glass,  by  means  of  a  curved 
pipette,  the  tube  containing  the  cotton  serving  as 
an  aspirator.  After  this  has  been  done,  the  second 
hole  is  stopped  with  a  cork. 

8almonsen's  Method  for  pure  cultures.  "An 
absolutely  pure  sowing  was  taken  from  putrefac- 
tion-specks in  defibrinized  ox-blood  which  had 
been  preserved  and  observed  in  capillary  tubes. 
In  order  to  obtain  as  large  a  number  as  possible  of 
different  forms,  he  chose  (1)  those  specks  which 
presented  the  greatest  possible  differences  in  refer- 
ence to  time  of  incubation,  rapidity  of  growth,  and 


76  THE  TECHNOLOGY   OF 

appearance  ;  (2)  specks  from  the  blood  of  different 
individuals  ;  and  (3)  he  employed  that  blood  only 
which  contained  only  a  small  number  of  specks, 
which  were  therefore  at  a  distance  apart.  The 
piece  of  capillary  tube,  the  contents  of  which  were 
going  to  be  sown,  was  then  separated  under  water 
by  a  strong  pair  of  scissors,  and  placed  in  the 
culture-bulb  with  the  requisite  precautions  — 
viz.,  using  all  the  instruments  immediately  after 
strong  heating,  to  destroy  the  dust,  etc.  A  bulb 
was  used  for  this  purpose,  provided  with  a  rather 
short  (4  cm.)  and  relatively  wide  neck,  with  only 
a  small  opening  closed  with  a  cap.  The  cap  was 
composed  of  a  rubber  tube,  which  was  so  firmly 
closed  for  half  its  length  by  a  small  wad-stopper 
that  it  was  slightly  bulged.  The  tube  was  some- 
what wider  than  the  upper  end  of  the  neck  of  the 
bulb,  that  it  might  be  placed  on  it  without  diffi- 
culty, but  narrower  than  the  lower  part.  After 
the  requisite  quantity  of  the  nutrient  fluid  had 
been  drawn  into  the  bulb,  and  the  latter  closed, 
the  definite  purification  and  sterilization  were 
effected  by  boiling."  (Journal  Micros.  Soc., 
1880,  p.  649.) 

LITERATURE    ON    CULTURE    METHODS. 

ALMQVIST  (E.)  :  "Die  besten  Methoden  Bacte- 
rien  rein  zu  cultiviren."  Botan.  Centralbl.  Bd. 
XIV.  1883,  p.  286. 


BACTERIA    INVESTIGATION.  77 

BIEDERT:  "Flatten  Culturen,"  Deutsche  med. 
Zeitung,  1884. 

BREFELD  :  I.  "  Kulturmethoden  zur  Unter- 
suchung  der  Pilze,"  Botanische  Untersuchungen 
uber  Schimmelpilze.  Hft.  IV.  (1881).  II.  "Die 
kunstlicher  Kultur  parasitischer  Pilze."  Botan. 
Unters.  iiber  Hefenpilze.  Bd.  V.  1883. 

ERMENGEN  (VAN)  :  "  Sur  les  methodes  de  cul- 
ture des  micro-organ i sines  pathogenes."  Bull.  Soc. 
Belg.  de  Microsc.  t.  IX.,  1883,  No.  8,  p.  105. 

FELEISEN  :  "  Ueber  neue  Methoden  d.  Unter- 
suchung  und  Cultur  pathogener  Bacterien."  Phys. 
med.  Gesellsch.  zu.  Wiirzburg,  1882,  p.  113-121. 

HEYDENREICH  (L.)  :  "Sur  la  sterilization  des 
liquids  au  moyen  de  la  marmite  de  Papin."  Compt. 
Rend.  T.  98,  p.  998  (1884). 

JOHNE  :  "  Ueber  die  Koch'sehen  Reinculturen." 
1885. 

KLEBS  (E.)  :  "  Ueber  fractionirte  Cultur"  in 
Archiv.  f.  experim.  Pathol.  Bd.  I,  p.  46,  also  Sitz- 
ber.  Phys.  med.  Gesellsch.  zu  Wiirzburg,  1873. 

KOCH  (R.)  :  "Flatten  kulturen."  Yertrag  auf 
dem  XI.  deutschen  Aerztetage,  1883,  zu  Berlin. 

PASTEUR  (L.) :  (Methods  of  culture  in  the  study 
of  alcoholic  fermentation).  Annales  de  Chemie  et 
de  Physique,  vol.  58,  p.  323. 

SALMON:  (On  a  New  Form  of  Culture  Tube). 
American  Monthly  Microscopical  Journal,  V. 
(1884).  — Journal  Roy.  Mic.  Soc.,  1885,  p.  145. 


78  THE  TECHNOLOGY  OF 

SALMONSEN  :  "  Zur  Isolation  differenter  Bak- 
terien  formen."  Bot.  Zeitung,  1876,  No.  39.  "Stu- 
dier  over  Blodets  Forraadnelse,"  1877.  "  Eine 
einfache  Methode  zur  Reinkulturen  verschiedener 
Faulnissbakterien."  Botau.  Zeitung,  1880.  No.  28. 

SMITH  (T.) :  "  Remarks  on  fluid  and  gelatinous 
media  for  cultivating  micro-organisms,  with  de- 
scription of  Salmon's  new  culture  tube,  and 
demonstration  of  the  process  of  using  it."  Amer. 
Monthly  Mic.  Journ.  V.  (1884),  p.  185. 

STERNBERG  (G.  M.) :  "  Methods  of  cultivating 
micro-organisms."  Report  Amer.  Assoc.  Adv. 
Sci.,  1881.  American  Monthly  Microsc.  Journ.  V. 
(1884),  pp.  183-185. 

III.  VACCINATION  OR  INOCULATION  EXPERIMENTS. 
In  inoculating  animals  with  the  pure  cultures  of 
pathogenic  Bacteria,  it  is  found  that  only  particular 
animals  are  susceptible  to  the  influence  of  certain 
microbes  ;  thus  birds,  especially  birds  of  prey,  are 
not  affected  by  the  Bacilli  of  anthrax ;  the  same 
little  rods  which  produce  a  fatal  disease  in  the 
domestic  mouse  are  harmless  to  birds,  rabbits, 
etc.  Even  animals  of  the  same  family  are  ex- 
empt; thus,  in  the  latter  case,  the  field  mouse  is 
unharmed  by  the  microbes  which  prove  fatal  to 
the  domestic  mouse.  The  mucous  surfaces  of 
mammals,  and  serous  surfaces  as  well  of  lower 
vertebrates  and  invertebrates,  are  not  suitable 


BACTERIA   INVESTIGATION.  79 

places  for  the  introduction  of  special  germs  whose 
action  on  the  system  it  is  desired  to  investigate, 
inasmuch  as  these  surfaces  are  invariably  inhabited 
by  a  great  variety  of  microbes,  as  we  have  already 
noticed.  The  serous  surfaces  of  birds  and  mam- 
mals, however,  are  not  in  normal  conditions,  so 
far  as  is  known,  inhabited  by  any  adult  microbes. 

TV'e  have,  therefore,  two  plans  by  which  the 
vaccination  or  inoculation  from  pure  cultures  may 
be  successfully  made.  After  having  removed  all 
hair  or  feathers  from  the  selected  spot,  and  having 
thoroughly  washed  the  skin,  and  bathed  it  with  an 
antiseptic  solution  (e.  g.,  two  per  cent  corrosive 
sublimate  solution),  dry  it  with  absorbent  cotton, 
and  make  a  cut  with  a  previously  sterilized  knife ; 
into  this  introduce  the  microbes  from  the  pure  cul- 
ture by  means  of  the  sterilized  platinum  wire.  If 
the  spot  is  now  covered  with  some  surgeon's  gauze 
or  cotton,  all  inoculation  from  the  air  may  be 
avoided.  The  pure  culture,  in  case  of  fluids,  may, 
by  means  of  a  sharp,  sterilized  canula,  be  injected 
at  once  into  the  mesodermal  tissues ;  an  ordinary 
hypodermic  syringe,  when  properly  sterilized, 
answers  the  purpose.  Any  special  modifications 
of  the  general  plan  will  be  noticed  in  the  appro- 
priate place  under  the  treatment  of  pathogenic 
Bacteria. 

Inoculation  methods  must  occupy  a  subordinate 
position  until  the  subject  of  attenuation  is  better 


80  THE  TECHNOLOGY  OF 

understood.  A  number  of  years  since,  Virchow 
(Archiv  fur  patholog.  Anat.  Phys.  Bd.  79,  p. 
213)  made  the  following  remark :  "  Truly,  we 
know  nothing  regarding  it  (the  question  of  im- 
munity), and  the  knowledge  of  Bacteria  has  also 
not  shed  the  least  light  upon  the  question."  That 
we  now  are  in  scarcely  better  position  is  indi- 
cated by  the  following,  taken  from  a  recent  article 
by  so  distinguished  a  physiologist  as  Lauder-Brun- 
ton  ("Practitioner"  for  September,  1884).  "To 
sum  up  the  results  of  these  laborious  investiga- 
tions, we  must  agree  with  Koch  and  Klein  that 
immunity  can  be  conferred  by  inoculation,  but 
this  immunity  can  only  take  rank  as  an  interesting 
theoretical  fact,  not  yet  brought  within  the  region 
of  practical  science.  We  must  deny  Pasteur's 
claim  that  his  method  of  animal  inoculation  gives 
absolute  immunity,  and  that  it  is  harmless,  the 
number  of  fatal  results  being  nil  or  very  small. 
There  is  no  doubt  that  when  Pasteur  performs  his 
inoculations  without  any  deaths,  he  is  working 
with  cultures  too  weak  to  give  any  immunity,  and 
also  that  in  many  cases  the  '  virulent '  anthrax 
Bacilli,  sent  out  by  him  as  a  test  for  the  protection 
already  conferred  by  vaccination,  are  much  too 
weak.  The  'vaccins,'  as  supplied  by  him,  have 
been  found  very  variable ;  thus,  sometimes  his 
*vaccin  premier1  has  killed  a  flock  of  sheep, 
whilst  'vaccin  deuxieme*  has  been  inert.  The 


BACTERIA    INVESTIGATION.  81 

objection  to  the  present  method  is  that  immunity 
can  only  be  conferred  by  a  percentage  of  losses 
from  death  greater  than  would  result  if  the  flock 
were  turned  upon  a  notoriously  infected  pasture, 
and  then,  further,  this  inoculation  favors  the  spread 
of  the  disease  by  the  formation  of  spores,  when 
any  of  the  Bacilli  fall  on  the  wool  of  the  animal ; 
also,  the  immunity  thus  given  lasts,  at  the  most 
favorable  computation,  no  longer  than  a  season. 
That  it  may  be  possible  in  the  future  to  discover 
a  method  of  attaining  immunity  without  too  great 
a  loss  during  the  process  must  be  allowed ;  but  to 
consider  it  proved  that  we  at  present  possess  such 
a  means,  in  the  method  of  inoculation  described  by 
M.  Pasteur,  can  only  lead  to  disappointment,  and 
to  a  monetary  loss  on  the  part  of  those  adopting  it." 

LITERATURE    ON    INOCULATION    METHODS. 

GRAWITZ  (P.) :  "  Die  Theorie  der  Schutzimp- 
fung;"  Yirchow's  Archiv.,  Ed.  84,  1883,  p.  87. 

HAY  (M.) :  "  Die  Technik  der  Vaccination  mit 
animaler  Lymphe,"  Wien  :  1881. 

KLEIN  (E.)  :  "  Micro-organisms  and  Disease." 
"Practitioner,"  London,  Oct.,  1884,  p.  248. 

SEMMER  (E.)  UNO  RAUPACH  (C.)  :  "  Beitrage  zur 
Lehre  von  der  Immunitat  und  Mitigation."  Dtsch. 
Zeitschr.  f.  Thiermedicin,  etc.,  VII.,  p.  347-363. 

STERNBERG  (Gr.  M.)  :  "  Vital  resistance  theory  of 
disease."  Amer.  Journal  of  the  Med.  Sci.,  Apr.  1, 


82  THE  TECHNOLOGY   OF 

1881.  II.  "  What  is  the  explanation  of  acquired 
immunity  from  infectious  disease."  (Abstr.  of  a 
paper  read  before  the  University  Scientific  Asso- 
ciation, Dec.  3,  1884.)  Johns  Hopkins  University 
Circular  for  January,  1885,  vol.  IV.  No.  36, 
p.  31. 

IV.  BIOLOGICAL  ANALYSIS. 

By  this  method,  answers  are  given  to  the  numer- 
ous questions  which  present  themselves  regarding 
the  various  phenomena  in  the  life  history  of  Bac- 
teria. The  results  of  investigation  so' far  tend  to 
contradict  the  generally  accepted  idea  that  Bacteria 
are  the  simplest  of  all  living  organisms.  Engel- 
mann  says  in  this  connection  :  "  Any  one  who  has 
closely  studied  the  form  and  mode  of  living  of  even 
the  most  common  forms  of  Bacteria  cannot  doubt, 
as  I  do  not  doubt,  that  these  organisms  are  rela- 
tively highly  organized,  and  can  only  be  consid- 
ered as  the  simplest  of  organisms  because  they 
are  the  smallest  which  we  can  know  ;  but  small 
and  simple  are  two  different  things.  .  .  .  Mor- 
phologically considered,  they  can  no  longer  be 
said  to  belong  to  the  simplest  organisms,  every 
Moner,  every  Plasrnodium,  stands  lower.  ...  I 
have  often  watched  the  separating  of  the  active 
bacterial  forms  from  the  stiff  Cladothrix  tree ;  it 
is  a  wonderful  sight,  and  I  do  not  know  with  what 
to  compare  it :  this  plant  at  the  moment  of  its 


BACTEKIA    INVESTIGATION.  83 

change  into  an  animal.  Physiologically  consid- 
ered, one  must  grant  that  they  possess,  like  true 
animals,  certain  powers  and  senses,  e.  g.,  the 
power  of  distinguishing  between  oxygen  and  car- 
bonic acid,  in  short,  a  capability  of  breathing ; 
and  further,  when  one  sees  how  they  swarm  about, 
and  consume  a  mass  of  nutriment  in  the  drop,  one 
must  grant  to  them,  in  addition,  another  sense, 
i.  e.,  the  need  of  nourishment.  There  are  also 
Bacteria  which  possess  a  specific,  and,  indeed,  a 
highly  organized  sense  for  light  and  color.  Of 
course,  by  these  phenomena,  they  are  elevated  far 
above  the  lowest  organisms,  above  all  typical 
plants ;  they  are,  physiologically  considered,  as 
far  as  they  show  the  above  and  similar  condi- 
tions, undoubtedly  animals,  and  it  is  only  so  much 
the  more  powerful  proof  of  the  unity  of  nature 
that  they,  through  their  morphological  develop- 
ment, extend  into  the  kingdom  of  plants." 

Zopf,  Koch,  and  others  indicate,  among  the 
questions  to  be  answered  in  a  thorough  study  of 
any  peculiar  Bacteria,  the  following  :  — 

1.  Shape,  size,  color,  and  details  of  structure, 
e.  g.,  flagella,  peculiar  envelope,  etc.? 

Character  and  speed  of  movements  ? 

2.  Character  of  natural  habitat? 

Artificial  media  best  adapted  to  growth  and 
reproduction  ? 

Stages  of  development  passed  through  ? 


84  THE  TECHNOLOGY  OF 

Formation  of  zoogloea,  spores,  filaments,  rods, 
cocci,  "swarms?" 

Conditions  under  which  such  formation  occurs  ? 

Character  of  colonies  formed  in  firm  culture 
media? 

3.  Capability  of  producing  fermentation?  putre- 
faction?     Character   of   decomposition   products, 
volatile  and  other,  formed  in  various    nourishing 
media? 

4.  Behavior    towards   oxygen   at   normal    and 
altered  pressures? 

Behavior  towards  other  gases  ? 

5.  Effects   of  various   temperatures   on   move- 
ment, germination,  etc.? 

6.  Behavior    in    relation    to    light    (phototonic 
properties)  ? 

Behavior  towards  electricity  ? 

7.  Behavior  towards  antiseptics  and  poisons? 

8.  Are  the  forms  under  investigation  found  in  a 
diseased  organ  or  tissue  ?     What  is  the  effect  of 
inoculating  animals  of  different  orders  and  species 
with  pure  cultures? 

If  virulent,  can  the  virulence  be  attenuated  by  ex- 
posure to  air,  to  antiseptics,  to  heat,  or  by  repeated 
"  fractional "  cultures  ?  Under  what  conditions  ? 

Does  the  inoculation  of  attenuated  germs  have  a 
cumulative  effect  if  repeated  at  short  intervals  ? 

Does  one  inoculation  give  immunity  towards  a 
second  made  with  virulent  microbes  ? 


BACTERIA    INVESTIGATION.  85 

Bacteria  may  be  counted  and  measured  by 
means  of  Professor  Thoma's  apparatus  for  count- 
ing blood  corpuscles,  as  made  by  Zeiss,  or  with 
an  ordinary  eye-piece  micrometer.  Szpilmann's 
method  for  studying  the  effect  of  gases  was  by 
the  use  of  a  Keeklinghausen's  moist  chamber,  with 
which  he  connected  two  tubes,  one  admitting  the 
gas,  the  other  allowing  of  its  escape.  The  tem- 
perature was  maintained  at  the  desired  point  by 
means  of  a  Valentine's  warm  table.  In  place  of 
the  above,  a  Ranvier's  gas-slide  may  be  used. 

Engdmann's  Method  for  studying  the  effect 
of  light  on  certain  Bacteria.  In  studying  the 
effect  of  light,  Engelmann  used  a  Sugg's  lamp, 
which  he  placed  in  a  dark  box  l  having  but  one 
round  opening ;  by  removing  all  diaphragms  he 
could  throw  an  image  of  the  lighted  opening  at 
any  point  in  the  field,  and  in  the  case  of  Bac- 
terium photometricum  found  that  within  a  few 
minutes  all  the  microbes  would  assemble  in  a 
dense  mass  at  the  lighted  point.  By  means  of 
the  thick  layer  obtained  in  this  way,  he  was  able 
to  subject  them  to  spectroscopic  examination,  to 
determine  their  color,  and  to  sliow  that  the  color- 
ing material  had  none  of  the  peculiar  characters 
of  chromophyll.  He  found  that  without  light 
there  was  no  movement,  and  that  with  the  return 

1  For  a  detailed  description  of  this  "  Mikroskopirkasten,"  vid. 
Pfluger's  Archiv.  f.  Physiol.  Bd.  XXIII.,  p.  577  (1880). 


86  THE   TECHNOLOGY  OF 

of  light  movement  began  anew,  a  condition  to  be 
compared  to  the  phototonus  of  higher  plants. 
Certain  other  Bacteria,  e.  g.  Spirillum  and  Bac- 
terium termo,  have  such  an  affinity  for  oxygen 
that  they  may  be  used  as  agents  for  detecting 
traces  of  free  oxygen  at  any  point  of  a  micro- 
scopic preparation,  and  by  means  of  this  Engel- 
mann  was  able  to  observe,  through  the  working  of 
different  parts  of  the  spectrum,  the  decomposition 
of  carbonic  acid  and  the  liberation  of  oxygen  by 
the  organisms  (in  the  case  of  Bacterium  chlori- 
num,  the  coloring  matter  of  which,  unlike  that  of 
Bacterium photometricum,  contains  chlorophyll) . 

Engelmann's  Method  for  the  determination  of 
a  chromophyll  assimilating  power  in  any  given 
Bacteria  by  means  of  other  Bacteria  used  as  re- 
agents for  the  detection  of  free  oxygen.  To  a 
drop  containing  as  large  a  number  as  possible  of 
the  Bacteria  to  be  examined,  add  a  very  small 
quantity  of  a  fluid  which  contains  numerous  speci- 
mens of  good,  lively  Bacterium  termo  or  Spiril- 
lum (the  latter  are  more  susceptible  reagents  for 
oxygen  than  the  ordinary  filth  Bacteria,  B.  termo), 
used  as  reagents  for  the  oxygen.  Seal  up  the 
preparation  with  some  vaseline.  After  ten  or 
fifteen  minutes  all  or  most  of  the  individuals  of 
B.  termo  or  Spirillum  will  come  to  a  rest  from  a 
lack  of  oxygen,  if  none  is  given  out  by  the  Bacteria 
under  investigation. 


BACTERIA    INVESTIGATION.  87 

Examine  points  where  the  Litter  have  assembled 
in  large  masses,  and  see  if  the  reagent  Bacteria 
have  gathered  about  these  masses  and  there  retain 
their  movements,  as  they  do  around  an  air  bubble 
or  a  green  cell. 

The  effects  of  antiseptics  and  poisons  upon  Bac- 
teria are  to  be  studied  by  adding  to  given  amounts 
of  culture  fluids  containing  the  living  microbes, 
certain  amounts  of  antiseptic  solutions  of  a  known 
strength,  varying  the  latter  in  a  regular  ratio 
through  a  series  of  experiments.  Electricity  and 
heat  may  be  applied  by  means  of  the  numerous 
simple  contrivances  described  in  every  manual  of 
histology  and  optician's  catalogue. 

Duclaux's  Method  for  studying  the  effect  of 
sunlight  upon  the  germs  of  Bacteria.  The  experi- 
ments were  made  with  Tyrothrix  scaber,  a  form 
which  flourishes  well  in  cultures  of  milk  and 
Liebig's  broth.  A  small  drop  of  a  pure  culture  was 
taken  at  the  moment  of  the  formation  of  spores, 
and  placed  in  the  bottom  of  a  small  culture  flask 
or  balloon,  closed  with  sterilized  cotton,  which 
allows  free  access  of  air,  but  not  of  new  germs. 
The  drop  evaporates,  the  flask  being  exposed  to 
the  summer  sun  upon  a  wall  for  fifteen  days,  one 
month,  or  two  months.  Other  flasks  prepared  in 
the  same  way  were  kept  in  an  oven,  where  they 
had  diffused  light  and  a  temperature  about  that  of 
the  maximum  obtained  from  the  sun.  At  the  end 


88  THE   TECHNOLOGY   OF 

of  the  periods  named,  culture  fluids  were  added  to 
the  dried  germs,  and  it  was  found  that  the  germs 
which  had  been  exposed  to  the  sunlight  had  lost 
their  vitality,  while  those  exposed  to  a  like  tem- 
perature, but  deprived  of  sunlight,  reproduced  and 
multiplied  very  rapidly. 

Pictet  and  Yung's  Method  for  ascertaining  the 
action  of  cold  upon  microbes.  The  various  cul- 
tures containing  numerous  lively  microbes  were 
hermetically  sealed  up  in  tubes,  which  were 
placed  in  a  wooden  box  enveloped  by  such  sub- 
stances as  are  poor  conductors  of  heat,  and  there 
kept  at  — 70°  C.  by  the  evaporation  of  liquid  sul- 
phurous acid,  or  by  means  of  solid  carbonic  acid, 
the  latter  being  constantly  renewed  by  means  of 
tubes  without  altering  the  pressure.  The  microbes 
were  kept  at  —70°  to  —  76°  C.  for  forty-eight 
hours,  allowed  to  stand  for  six  hours  at  ordi- 
nary temperatures,  and  then  again  frozen.  The 
degree  of  temperature  was  determined  by  means 
of  the  formula  given  by  MM.  RaouV  Pictet,  and 
Cellerier  in  their  Memoire  upon  the  maxima  ten- 
sions of  saturated  vapors. 

LITERATURE    OF    THE    BIOLOGY    OF    BACTERIA. 

BECHAMP  (A.)  :  "  Les  Microzymas  gastriques  et 
la  pepsine."  Compt.  Rend.  T.  XCIY.,  p.  970. 

BLACK  (G.  V.) :  "  The  formation  of  poisons  by 
micro-organisms,  a  biological  study  of  the  germ 


- 

BACTERIA    INVESTIGATION.*-  89 

theory    of    disease."        Philadelphia    Blakiston), 
1884,  12mo. 

BOEHLENDORFF  (H.  v.)  :  "  Ein  Beitrag  zur  Bio- 
logie  einiger  Schizomyceten."  Inaug.  Dissert. 
Dorpat,  1880. 

BRIEQER  (L.)  :  "Ueber  Spaltungsproducte  der 
Bakterien. '  Zeitschr.  f.  phys.  Chemie,  VIII., 
p.  306. 

BRISSON  (T.) :  (Fatty  bodies  as  generators  of 
Bacteria).  Untersuch.  iiber  niedere  Pilze  aus  dem 
pflanzen  physiol.  Institut.  Miinchen,  I.  (1882), 
p.  129-159.  —  Journ.  Roy.  Mic.  Soc.,  ser.  II.,  vol. 
Ill,  p.  106. 

BUCHOLTZ  (L.)  :  I.  "Antiseptica  und  Bacte- 
rien."  Archiv.  f.  experim.  Pathol.  Bd.  IV. 
1875.  II.  "Ueber  das  Verhalten  von  Bacterien 
zu  einige  Antisepticis.  Inaug.  Dissert."  Dorpat, 
1876. 

CERTES  (A.):  "De  Faction  des  hautes  pres- 
sions  sur  le  phenomenes  de  la  putrefaction  et  sur 
la  vitalite  des  micro-organismes  d'eau  douce  et 
d'eau  de  mer."  Compt.  Eend.  T.  99.  25  Aug. 
1884.  p.  385. 

CHAIRY  :  "  Action  des  agents  chimique  puis- 
santes  sur  les  bacteries  du  genre  Tyrothrix  et  leur 
spores."  Ibid.  T.  99.  Dec.  1,  1884.  p.  980. 

CHAPPUIS  (E.) :  (Action  of  ozone  on  germs 
contained  in  the  air).  Bull.  Soc.  Chim.  XXXV., 
p.  390.— -Journ.  Chem.  Soc.  (abstr.),XI.  (1881), 


90  THE  TECHNOLOGY  OF 

p.  632.  Journ.  Roy.  Mic.  Soc.  (ii),  vol.  L, 
p. 781. 

DE  LA  CROIX  (N.  J. )  :  "  Das  Verhalten  der 
Bakterien  des  Fleischwassers  gegen  einige  Anti- 
septica."  Archiv.  f.  experim.  Pathol.  Bd.  XIII. 
H.  3.  p.  175. 

DUCLAUX  (E.)  :  "Influence  de  la  lumiere  du 
soleil  sur  la  vitalite  des  germes  de  microbes." 
Compt.  Rend.  T.  100.  No.  2.  Jan.  12,  1885. 
p.  119. 

ENGELMANN  (Tn.  W.)  :  I.  "  Untersuchungen 
iiber  d.  quantitat  Bez.  zw.  Absorption  des  Lichtes 
und  Assimilation  (microspektral  Photometer)." 
Botan.  Zeitung,  No.  6,  7.  1884.  II.  "Zur  Bio- 
logie  der  Schizomyceten."  Pfliiger's  Archiv.  f. 
Physiol.  Bd.  26.  p.  537.  III.  "Bacterium 
pkotometricum."  Ibid.  Bd.  30.  p.  95.  1882. 
Taf.  1.  IV.  (Describes  his  dark  '  Mikrosko- 
pirkasten')  ibid.  (1880),  p.  577.  V.  (Bacterial 
investigation  of  sunlight,  gaslight,  and  the  light  of 
Edison's  lamp.)  K.  Accad.  Wetensch.  Amsterdam, 
Nov.  25,  1882.— Botan.  Centralbl.  XIII.  (1883), 
p.  214.  Journ.  Roy.  Mic.  Soc.,  Ser.  ii.,  vol.  III., 
p.  401.  VI.  "Neue  Methode  zur  Untersuchung 
der  Sauerstoffansscheidung  pflanzliche  und  thier- 
ischer  Organismen."  Bot.  Zeitung,  1881,  No.  28. 
VII.  "Farbeund  Assimilation."  Ibid.  1883.  No. 
1.  p.  2. 

FORBES    (S.  A.):      "The    use   of    contagious 


BACTERIA    INVESTIGATION.  91 

germes  as*  insecticides."  Amer.  Naturalist,  1883, 
p.  1169. 

GAUTIER  :  "  Sterilization  a  froid  des  liquids 
fermentiscibles."  Bulletin  de  la  Socie'te  Chemique, 
1884,  vol.  42,  p.  146. 

GAYON  (U.)  :  (Crystallizable  substances  pro- 
duced by  a  bacterium).  Bull.  Soc.  Bot.  France, 
XXVIII.,  p.  321. 

GROHMANN  (W.)  :  "TJeber  die  Einwirkung  des 
zellenfreien  Blutplasrna  auf  einige  pflanzliche  Mi- 
kroorganismen  "  (Schimmel —  spross —  pathogene 
u.  nicht  pathogene  Spaltpilze),  Dorpat.  1885  (C. 
Kruger),  gr.  8. 

GROSSMANN  u.  MAYERHAUSEN  :  (Effects  of 
Oxygen  and  Hydrogen  on  Bacteria).  Pfliiger's 
Archiv.  Bd.  XV.,  1877.  p.  245. 

HABERKORN  (Tn.)  :  "Das  Verhalten  von  Harn- 
bakterien  gegen  einige  antiseptica."  Inaug.  Dis- 
sert. Dorpat,  1879. 

HAMLET  (WM.)  :  "Action  of  Compounds  Inim- 
ical to  Bacterial  Life."  Journ.  Chem.  Soc., 
XXXIX.  (1881),  p.  326-331. 

HOPPE-SEYLER  (F.)  :  (Action  of  Oxygen  on 
Low  Organisms).  Zeitschr.  f.  physiol.  Chem. 
VIII.  (1884),  p.  214.— Naturforscher,  XVII. 
(1884),  p.  116. 

HUEPPE  :  "  Ueber  einige  Vorfragen  zur  Desinfec- 
tionslehre  und  iiber  die  Hitze  als  Desinfectionsmit- 
tle."  Deutsche-militararztl.  Zeitschr.  1882.  No.  3. 


92  THE  TECHNOLOGY   OF 

JAMIESON  (J.)  :  "  The  influence  of  light  on  the 
development  of  Bacteria."  Nature,  1881.  vol. 
XXV. 

KOCH,  GAFFKY,  AND  LCEFFLER  :  "  Versuche 
iiber  die  Verwerthbarkeit  heisser  Wasserdampfe 
zu  Desinfectionszwecken."  Mitt.  a.  d.  kais.  Ge- 
sundheitsamt.  I.,  1881,  p.  322. 

KUHN  (P.)  :  "  Ein  Beitrag  zur  Biologie  der 
Bakterien."  Inaug.  Dissert.  Dorpat.  1879. 

LUDWIG  (F.)  :  "Ueber  die  spectroscopische 
Untersuchung  photogener  Pilze  (Micrococcus 
pflugeri)"  Zeitschr.  f.  wiss.  Microsc.  Bd.  I. 
H.  2.  p.  181. 

MELSENS  ;  "Reclamation  de  priorite,  a  propos 
de  communications  recents  sur  la  vitalite  des  virus 
et  de  la  levure  de  biere."  Compt.  Rend.  T.  98, 
p.  923. 

MEYER  (VAN  OVERBECK  DE — )  :  "Ueber  den 
lahmenden  Einfluss  von  Sauerstoff  sehr  hoher 
Spannung  auf  Bacterien  "  in  das  Onderzoekingen 
ged.  in  het.  physiol.  labor.  Utrecht.  Derde.  R. 
VI.,  1881,  p.  151-196. 

MIQUEL  ET  BENOIST  :  "  Sterilization  a  froid." 
Bull,  de  la  Soc.  Chem.  de  Paris  (1881),  vol.  35, 
p.  552. 

MIQUEL  (P.):  "  Les  organismes  vivant  de 
I'atmosphere."  Paris,  1883.  Chapt.  IX.  "Des 
substances  Antiseptiques." 

NAEGELI  (C.  v.)  :  I.  "  Untersuchungen  iiber  nie- 


BACTERIA    INVESTIGATION.  93 

der  Pilze,"  a  us  dem  pflanzen-physiolog.  Inst.  in 
Miinchen.  (R.  Oldenberg.)  1882.  II.  "Ueber 
die  Bewegungen  kleinster  Korperchen."  Miin- 
chener  Acad.  Sitzungsber,  math-phys.  Kl.  1879, 
p.  389.  III.  "  Ernahrung  der  niederen  Pilze 
durch  Kohlenstoff  und  StickstoftVerbindungen." 
Untersuchungen  iiber  niedere  Pilze,  1882. 

NENCKI  UND  LACHOWICZ  :  "Die  Anserobiose- 
frage."  Archiv  f.  d.  ges.  Physiologic.  Bd. 
XXXIII.  (1883). 

NENCKI  u.  SCHAFFER  :  "  Ueber  die  chemische 
Zusammensetzung  der  Faulnissbacterien."  Journ. 
f.  pract.  Chem.  N.  F.  XX.,  p.  443. 

PATOUILLARD  (N.):  (Phosphorescence  caused 
by  Bacteria).  Revue  Mycol.  IV.  (1882),  p.  208- 
209,  1  pi. — Journ.  Roy.  Mic.  Soc.,  Ser.  II.,  vol. 
in.,  p.  106. 

NENCKI  (M.)  :  "  Beitr  ge  zur  Biologic  der 
Spaltpilze,"  mit  2,  lith.  Tan.  Leipzig.  1880. 

PICTET  ET  YUNG  ;  "  De  Faction  du  froid  sur 
les  microbes."  Compt.  Rend.  T.  98,  p.  467. 
1884. 

REINKE  (J.)  :  (Influence  of  Concussion  on  the 
growth  of  Bacteria).  Pfliiger's  Archiv.  f.  Physiol. 
XXIII.,  p.  434.  Naturforscher,  XIV.,  1881,  p.  56. 

RICHET  (C.)  :  "De  1'action  toxique  compared 
des  me'taux  sur  les  microbes."  Compt.  Rend.  T. 
XCVII.  (1883),  p.  1004-1006.  cf.  Journ.  Roy. 
Mic.  Soc.,  1884,  p.  427. 


94  'HE  TECHNOLOGY   OF 

SCHNETZLER  (J.  B.)  :  "  De  Faction  du  Curare 
sur  les  fibres  musculaires,  les  cils  vibratils  et  les 
Bacteries,  contribution  a  1'etude  des  Bacteries." 
Bull.  Soc.  Yaudois  Sci.  Nat.  XVII.  (1881),  p. 
625-632. 

SCHWARTZ  (N.)  :  "  Ueber  das  Verhalten  einiger 
Antiseptica  zu  Tabacksinfusbacterien."  Pharma- 
ceutische  Zeitschr.  f.  Russland,  1880. 

THIN  (G.)  :  "On  the  absorption  of  pigment  by 
Bacteria."  Proc.  Roy.  Soc.,  London,  vol.  31 
(1881),  p.  503-504. 

TIEGHEM  (P.  VAN)  :  (Bacteria  living  at  high 
temperatures).  Bull.  Soc.  Bot.  de  France, 
XXVIII.  (1881),  p.  35,  36. 

VANDEVELDE  (G.)  :  "  Studien  zur  Chemie  des 
Bacillus  subtilis."  Zeitschr.  f.  phys.  Chemie. 
VIII.,  p.  367. 

WOSNESSENSKI  ( J. )  i  "Influence  de  1'oxygene 
sous  pression  augmentee  sur  la  culture  du  Bacillus 
Anthracis."  Compt.  Rend.  T.  98,  p.  314. 
(1884.) 

WERNICH  (A.)  :  "Die  aromatischen  Faulniss- 
producte  in  ihre  Einwirkung  auf  Spalt-  und 
Sprosspilze."  Virchow's  Archiv.  f.  path.  Anat., 
etc.  Bd.  78.  p.  54. 

WORTMANN  (J.)  :  (Diastatic  Ferment  of  Bac- 
teria). Zeitschr.  f.  physiol.  Chemie.  VI.,  p.  287. 
Naturforscher,  XV.  (1882),  p.  321. 


BACTERIA  INVESTIGATION. 


PART  II. 

SPECIAL  METHODS   FOR   INVESTIGATING 
PARTHOGENIC   BACTERIA. 

ANY  discussion  as  to  the  r61e  played  by  Bacteria 
in  disease  is  here  out  of  place :  whether  they  are 
simple  concomitants,  the  Bacteria  of  health  taking 
advantage  of  a  lowered  vitality  to  develop  with 
unwonted  vigor  and  rapidity,  thus  abetting  the 
disease  by  depriving  the  tissues  of  the  materials 
required  for  their  support  (e.g.,  oxygen,  pabulum) , 
as  indicated  by  Pasteur  for  the  microbe  of  chicken 
cholera  :  "  On  peut  aisement  le  comprendre.  Le 
microbe,  par  example,  est  aerobie  ;  il  absorbe  pen- 
dant sa  vie  de  grandes  quantities  d'oxyge*ne,  et  il 
brule  boucoup  des  princeps  de  son  milieu  de  cul- 
ture, ce  dont  il  est  facile  de  s'assurer  en  comparent 
les  extraits  du  bouillon  de  poule  avant  et  apres  la 
culture."  Whether  they  bring  about  death  or 
local  disturbance  by  interfering  mechanically  with 
the  vital  functions  as  emboli,  or  whether  they  are 
simple  carriers  of  the  infectious  material,  alcaloid, 
zymase  or  ptomaine,  or  produce  them  by  their 
activity  or  by  chemical  changes  which  occur  after 
they  have  finished  their  life  history,  as  Dr.  Freire 

95 


96  THE   TECHNOLOGY  OF 

thinks  is  the  case  in  yellow  fever,  whether  they 
are  in  themselves  the  specific  materies  morbi  or 
not,  are  questions  to  which  we  can  only  refer  in 
passing  to  the  real  intent  of  this  little  book,  which 
is  to  place  before  investigators  the  means  of  answer- 
ing these  questions  for  themselves.  That  Bacteria 
are  present  in  disease,  as  well  as  in  health,  cannot 
be  disputed ;  that  they  often  seem  to  be  the  cause 
of  death,  if  not  the  exciting  cause  of  the  disease, 
is  frequently  remarked.  By  the  following  pages  it 
will  be  seen  that  many  specific  forms  have  been 
described.  The  reasons  for  doing  so  are  in  some 
cases  apparently  well  founded,  while  others  need 
to  be  further  investigated,  and  more  testimony 
brought  to  bear.  In  this  connection  Dr.  Koch 
briefly  characterizes  his  position  as  follows  :  — 

"  It  is  not  yet  proven  that  all  infectious  diseases 
arise  through  parasitic  micro-organisms,  and  on 
this  account  the  parasitic  character  of  the  disease 
must  in  every  case  be  proven.  The  first  step  to 
this  knowledge  rests  upon  the  careful  investigation 
of  all  portions  of  the  body  altered  by  the  disease, 
to  render  certain  the  presence  of  parasites,  their 
distribution  in  the  diseased  organs,  and  their  rela- 
tions to  the  tissues  of  the  body.  It  is  apparent 
that  all  helps  which  are  offered  by  modern  micro- 
scopical technology  should  be  brought  into  use. 
The  tissues  and  the  tissue  juices,  the  blood,  lymph, 
etc.,  fresh,  with  and  without  reagents,  are  to  be 


BACTERIA    INVESTIGATION.  97 

examined  microscopically.  They  are  dried  upon 
the  cover-glass,  and  handled  according  to  the  dif- 
ferent processes  of  staining.  The  hardened  objects 
can  be  cut  into  sections  by  means  of  the  microtome, 
then  stained,  and  the  microscopic  preparations 
thus  obtained  subjected  to  a  penetrating  examina- 
tion by  means  of  suitable  illumination  and  with 
the  best  objectives.  Only  after  a  thorough  inves- 
tigation has  been  made  in  this  way,  to  ascertain 
whether  micro-organisms  are  to  be  found  in  dis- 
eased parts,  in  what  organ  they  are  to  be  found  in 
the  greatest  purity,  whether,  for  example,  in  the 
lung,  spleen,  blood,  etc.,  can  an  effort  be  made  to 
arrive  at  a  conclusion  as  to  whether  they  are  of  a 
pathogenic  nature  and  the  special  cause  of  the 
existing  disease.  For  this  purpose  they  are  then 
raised  in  pure  cultures,  and  when  they  are  by  this 
means  freed  from  all  the  originally  adhering  ele- 
ments of  the  diseased  body,  they  are  inoculated 
back  on  the  same  species  of  animal  if  possible,  or 
on  some  animal  which  will  show  unmistakably  the 
same  symptoms.  In  order  to  illustrate  this,  I  recall 
tuberculosis.  First,  it  is  esta-blished,  by  micro- 
scopical investigation  with  the  aid  of  staining 
reagents,  that  there  are  strongly  characteristic 
Bacilli  present  in  the  diseased  organs ;  these 
Bacilli  are  then  isolated  in  pure  cultures,  in  such 
a  manner  that  they  are  not  mixed  with  other 
Bacilli  and  thereby  made  impure,  and  are  next 


98  THE  TECHNOLOGY   OF 

inoculated  back  on  as  many  animals  as  possible  of 
such  different  kinds  as  are  known  to  be  susceptible 
to  this  disease.  The  disease  is  engendered  anew. 
Another  very  good  illustration  is  erysipelas  of  man. 
It  has  been  long  known  that  Micrococci  are  con- 
stantly to  be  found  in  the  lymph  vessels  of  the  skin 
in  this  disease  ;  this,  however,  did  not  prove  by  far 
that  they  were  the  cause  of  the  disease.  But  since 
Fehleisen  has  succeeded  in  making  cultures  from 
pieces  of  skin  cut  from  a  person  suffering  from 
erysipelas,  purifying  from  the  possible  presence  of 
other  germs  on  the  surface  by  cauterization,  and 
after  breeding  the  Micrococci  in  pure  cultures,  pro- 
ducing a  typical  erysipelas  again  upon  other  men 
through  inoculation,  there  can  be  no  further  doubt 
that  Micrococci  are  the  cause  of  erysipelas,  and  it 
is  to  be  regarded  as  a  parasitic  disease." 

The  following  remarks  of  M.  Pasteur  may  not 
be  out  of  place  just  here,  suggesting  as  they  do 
the  propriety  of  carefully  avoiding  any  unneces- 
sary additions  to  the  specific  nomenclature  of  Bac- 
teria. "  Qu'il  me  soit  per  mis  d'aj  outer  que,  dans 
mes  Communications  concernant  les  ormmismes 

o 

microscopiques,  je  me  suis  abstenu  generalement 
de  donner  des  noms  specifiques  a  ceux  de  ces 
organismes  que  je  pouvais  croire  nouveaux.  Si 
cela  etait  necessaire,  il  est  toujours  preferable  tie 
caracteriser  ces  petits  etres  par  une  ou  plusieurs 
de  leurs  fonctions.  Autant  les  denominations 


BACTERIA  INVESTIGATION.  99 

speciales  sont  utile  et  commodes  quand  on  les 
appliques  a  des  etres  bien  connus,  autant  elles 
peuvent  creer  d'embarras  et  de  confusion  lorsqu'il 
s'agit  d'organismes  tres-voisins  par  leurs  formes  et 
qui  peuvent  etre  tres-dissemblables  par  leurs  pro- 
prietes  physiologiques."  1 


ANTHRAX. 

(Malignant  pustule,  Splenic  fever.)     Bacillus  anthracis 
(Colin). 

Toussaint's  Method. — In  1880,  M.  Toussaint, 
Professor  in  the  Veterinary  School  in  Toulouse,  an- 
nounced to  the  Academy  of  Sciences  of  Paris  that 
he  had  discovered  a  process  by  which  he  could,  by 
inoculating  animals  with  an  artificially  weakened 
anthrax  poison,  give  them  an  entire  immunity  to- 
wards further  effects  of  this  poison.  His  method 
of  reducing  the  virulence  of  the  poison  was —  (1) 
to  place  some  of  the  defibrinated  blood  of  an  ani- 
mal suffering  from  anthrax,  for  about  ten  minutes, 
under  the  influence  of  a  temperature  of  55°  C., 
using  it,  after  cooling,  for  inoculation;  or  (2)  to 
treat  anthrax  blood  with  about  one  quarter  per 
cent  of  carbolic  acid,  and  use  this  mixture  for  in- 
oculation. He  believed  that  by  these  methods  the 
Bacilli  were  killed,  without  at  the  same  time  de- 
stroying the  infectiousness  of  the  material  con- 

1  Compt.  Rend.  T.  88  (1879),  p.  1217. 


100  THE  TECHNOLOGY   OF 

cerned,  but  only  rendering  it  milder.  Loeffler, 
however,  proved  this  idea  to  be  fallacious,  and 
experiments  were  made  by  Bouley,  Chaveau,  and 
others,  but  it  was  not  until  Feb.  28,  1881,  that  the 
proper  method  of  treating  the  Bacilli  was  made 
public. 

At  this  time  the  Academy  of  Paris  received  the 
announcement  of  — 

Pasteur's  Method,  by  which  he  was  able,  through 
artificial  culture,  to  produce  a  lymph  which  could 
be  used  without  danger  as  a  protective  vaccination 
against  anthrax.  This  weakening  of  the  virulence 
of  the  microbes  was  brought  about  by  exposing 
them  to  the  influence  of  atmospheric  air  under  cer- 
tain conditions,  and  he  had  successfully  vaccinated 
sheep,  rabbits,  and  guinea-pigs.  He  found  by 
experiment  that  anthrax  Bacilli  develop  best  at 
a  temperature  of  from  25°  to  40°  C.  ;  at  higher  or 
lower  temperatures  the  increase  is  lessened,  stop- 
ping entirely  at  15°  C.  as  well  as  at  45°  C. 

It  may  here  be  briefly  explained  that  the  anthrax 
Bacilli  possess  three  developmental  forms — (1) 
little  rods  (Bacteridia)  ;  (2),  spherical,  spore-like 
bodies,  called  "  resting  spores  ;"  and  (3),  minute 
glistening  granules  (Yirchow  and  Ruloff).  In  the 
"resting"  spore  stage  they  exhibit  a  great  power 
of  resistance  towards  external  influences.  Pasteur 
investigated  anthrax  Bacilli  contained  in  blood, 
at  a  temperature  of  42°-43°  C.,  in  a  culture-  fluid 


BACTERIA    INVESTIGATION.  101 

of  beef  broth.  He  studied  the  effect  of  exposing 
them  to  the  influence  of  filtered  atmospheric  air ; 
this  he  accomplished  simply  by  stuffing  both  open- 
ings to  the  culture  apparatus  with  cotton,  allowing 
the  gases  of  the  atmosphere  to  pass,  but  excluding 
any  contained  organic  germs.  He  found  that 
under  these  conditions  the  Bacilli  lost  their  viru- 
lence to  such  an  extent  that  he  could  in  the  course 
of  fourteen  days  vaccinate  sheep  without  danger. 
In  cases  where  the  culture  had  only  remained  for 
twelve  days,  one  half  of  the  animals  vaccinated 
died.  In  the  cultures  held  at  42° :  43°  C.  the  Ba- 
cilli retained  their  vitality  for  from  four  to  six 
weeks,  and  were  useful  for  vaccination  during  that 
time  ;  after  that,  they  died.  He  found  that  if  sheep 
which  had  been  vaccinated  with  a  culture  fluid, 
twenty-four  days  old,  containing  living  anthrax 
Bacilli,  were  again  vaccinated  within  twelve  days 
with  Bacilli  taken  direct,  and  in  their  full  virulence, 
from  a  sick  animal,  they  died;  but  if  this  second 
vaccination  was  made  with  a  twelve  days'  old  cul- 
ture, the  sickness  following  was  slight,  and  the  sheep 
were  afterwards  found  to  be  fully  protected  against 
vaccination  with  the  most  virulent  germs.  The 
second  vaccination  should  not,  in  any  case,  be  made 
before  twelve  days  have  elapsed,  as  the  effect  seems 
to  be  cumulative,  and  death  will  follow  in  many 
cases.  Time  enough  must  be  allowed  for  the  first 
sickness  to  pass  off.  Pasteur  next  proceeded  as 


102  THE  TECHNOLOGY   OF 

follows  to  make  a  culture  which  should  be  good 
for  more  than  six  weeks.  Take  from  the  culture, 
during  the  first  six  weeks,  a  drop,  and  convey  it 
to  sterilized  meat  broth  which  is  maintained  at  a 
constant  temperature  of  35°  C.  The  Bacilli  will 
increase  rapidly  here,  and  the  newly-resulting  gen- 
eration will  possess  exactly  the  same  grade  of  viru- 
lence which  was  possessed  by  the  organisms  of  the 
first  culture  at  the  moment  the  drop  was  removed. 
After  forty-eight  hours  the  second  culture  begins 
to  form  "  resting  spores,"  and  becomes  entirely  in 
this  condition  in  the  course  of  a  few  days.  These 
anthrax  spores  now  possess  the  same  degree  of 
virulence  as  the  rods  at  the  time  of  removing  the 

O 

drop.  In  this  manner  anthrax  cultures  may  be 
made  of  any  chosen  virulence,  which,  in  hermeti- 
cally sealed  glass  tubes,  will  retain  their  efficiency 
for  a  year  or  more.  A  culture  fluid  of  this  kind 
(anthrax  lymph)  can  be  used  as  a  vaccinating  ma- 
terial, or  for  making  new  cultures  in  fresh  sterilized 
broth.  Pasteur  named  the  twenty-four  day  old 
culture,  as  well  as  the  secondary  brood  made  from 
it,  the  first  vaccine  ("premier  vaccin,"  ersten  Imp- 
stoff) ,  because  he  used  it  for  his  first  protective  vac- 
cination. It  is  comparatively  harmless  in  large 
amounts  for  rabbits,  guinea-pigs,  sheep,  and  other 
larger  domestic  animals,  while  it  will  still  kill 
mice. 

The  twelve-day  old  culture,  and  that  of  its  sec- 


BACTERIA    INVESTIGATION.  103 

ondary  brood,  Pasteur  called  the  second  vaccine 
("deuxieme  vaccin,"  zweiten  ImpstofF),  because  it 
served  him  for  a  second  vaccination  twelve  days  after 
the  first.  Twelve  days  after  this  last,  there  should 
be,  as  a  rule,  absolute  immunity  towards  anthrax. 
Pasteur  and  his  assistants,  Chamberland  and  Roux, 
usually  vaccinate  animals  upon  the  ear  or  on  the 
thinly-haired  inner  surface  of  the  thigh,  or  inject 
the  culture  under  the  skin  with  a  hypodermic 
syringe.  Cattle  are  found  to  be  less  easily  pro- 
tected by  vaccination  than  sheep.  In  eighty  per 
cent  of  the  latter,  immunity  is  known  to  extend 
over  a  year,  and  perhaps  for  a  lifetime. 

Chaveau's  Method.  Toussaint's  method  was  to 
attenuate  the  virulence  of  the  anthrax  microbes  by 
heat  applied  to  the  germs  while  in  the  fluids  of 
the  diseased  animal.  Chaveau  made  an  improve- 
ment upon  this,  and  also  demonstrated  that  the 
presence  of  oxygen,  as  supposed  by  Pasteur,  has 
nothing  to  do  with  the  attenuation.  He  placed 
the  Bacilli  under  conditions  where  they  were  more 
susceptible  to  heat.  He  inoculated  sterilized  broth 
with  fresh  anthrax  blood,  placed  the  flask  in  an 
oven,  and  maintained  the  temperature  at  42°-43° 
C.,  but  instead  of  keeping  the  culture  here,  as  in 
Pasteur's  method  of  attenuation,  for  twelve  or 
thirteen  days,  he  removed  the  flask  after  about 
twenty  hours,  and  placec^  it  in  another  oven,  at 
47°  C.  ;  for  one,  two,  three,  or  four  hours.  This 


104  THE  TECHNOLOGY   OF 

terminated  the  process ;  it  did  not  destroy  the 
virulent  agents,  but  they  were  found  to  have  lost 
more  or  less  of  their  noxious  property.  He 
treated  cultures  which  had  had  all  air  removed 
by  means  of  a  force-pump,  with  equally  favorable 
results. 

Chaveau's  Method  for  the  preparation  and  atten- 
uation of  large  cultures.  Two  periods  are  required  : 
first,  for  the  preparation  and  development  of  the 
attenuated  germs;  second,  for  the  complementary 
attenuation  of  the  spores  which  result.  (1.)  A 
drop  of  fresh  infectious  blood  is  taken  from  a 
diseased  animal  and  placed  in  a  glass  culture-flask, 
containing  twenty  grammes  of  sterilized  broth, 
and  retained  for  two  hours  at  43°  C.,  and  then 
heated  for  three'  hours  at  47°-49°  C.  (2.)  Glass 
chemical  flasks,  having  three  openings  and  a  ca- 
pacity of  one  or  two  litres,  will  furnish  sufficient 
culture  virus  for  the  inoculation  of  from  four  to 
eight  thousand  sheep.  Fill  the  flasks  five  sixths 
full  of  sterilized  broth.  The  middle  opening  is 
furnished  with  a  long  tube,  descending  to  the 
bottom  of  the  flask.  It  is  by  means  of  this  tube, 
whose  exterior  extremity  is  plugged  with  cotton, 
that  air  is  introduced  in  fine  bubbles.  Of  the  two 
lateral  openings,  one  gives  rise  to  an  adductor 
tube ;  the  other  is  drawn  out  into  a  slender  tubule 
for  emptying  the  small  flasks.  It  is  by  means  of 
this  last  slender  tube,  that,  aspirating  on  the  sec- 


BACTERIA    INVESTIGATION.  105 

ond  tube,  we  introduce  the  prepared  microbes,  in 
proportion  of  one  drop  to  ten  grammes  of  culture 
fluid,  or  eight  grammes  of  seed  fluid  to  a  culture 
of  sixteen  hundred  grammes,  which  may  be 
doubled  or  tripled  if  the  seed  is  poor.  After 
the  introduction  of  the  seed  fluid,  the  slender 
tube  is  closed  by  fusion  in  the  lamp  flame.  The 
large  culture  thus  prepared  is  placed  in  a  thermo- 
stat at  35°-37°  C.  The  development  is  incomplete 
if  it  is  allowed  to  remain  at  rest ;  but  when  air  is 
caused  to  pass  through  it,  by  means  of  the  aspira- 
tor, it  proliferates  abundantly.  In  one  week  the 
evolution  is  usually  terminated,  and  a  rich  forma- 
tion of  spores  is  found,  attenuated  by  the  heat. 
The  culture  fluid  best  adapted,  in  Chaveau's  opin- 
ion, is  chicken  broth  (one  part  meat,  four  to  five 
parts  water).  The  current  of  air  should  be  very 
regular,  and  traverse  the  culture  fluid  in  quantities 
of  about  one  or  one  and  a  half  litres  per  hour. 
Shake  the  flasks  carefully,  night  and  morning. 
The  nearer  the  temperature  is  kept  to  40°  C.  the 
better  the  culture.  (3.)  From  a  large  flask  fill  a 
dozen  of  the  little  tubes  used  by  Pasteur  for  the 
distribution  of  vaccine,  and  place  some  of  these 
in  a  water-bath,  others  in  an  air-bath,  and  heat 
carefully  up  to  89°-90°  C.  Watch  carefully,  and 
note  the  degree  of  temperature  at  which  all  power 
of  proliferation  is  destroyed.  Now  for  the  vaccine 
to  be  used  first  ("premier  vaccin")  heat  the  cul- 


106  THE  TECHNOLOGY   OF 

tures  to  the  nearest  possible  point  to  that  which 
deprives  them  of  proliferating  power ;  for  the 
second  vaccine  ("deuxieme  vaccin")  heat  to  a 
point  two  degrees  less  than  the  first.  Each  time 
a  large  culture  is  made,  the  degree  of  this  final 
heating  must  be  ascertained ;  usually  84°  C.  an- 
swers for  the  first,  and  82°  C.  for  the  second. 
Sometimes  80°  C.  has  given  a  good  premiere 
vaccin,  and  78~  C.  a  second.  Cultures  placed 
under  the  same  conditions  will  not  always  give 
the  same  degree  of  attenuation  in  their  viru- 
lence. Use  a  large  amount  of  water  in  the 
water-bath,  and  have  the  regulator  so  arranged 
that  the  heat  does  not  vary  from  the  required 
temperature. 

Chamberland  and  Roux's  Method.  To  over- 
come the  objection  to  Chaveau's  method,  that  the 
cultures  made  with  his  attenuated  microbes  did  not 
retain  their  attenuation,  Chamberland  and  Roux 
proceeded  to  modify  Toussaint's  second  method. 
They  introduced  into  beef  broth  cultures  of  Ba- 
cillus anthracis  which  had  been  neutralized  with 
potash,  variable  quantities  of  antiseptics,  and 
placed  the  cultures  in  an  oven  at  35°  C.  The 
flocculent  growth  of  the  Bacteria  wras  greatest 
where  the  least  antiseptic  had  been  used,  gradu- 
ally diminishing  as  the  percentage  of  antiseptic 
increased  up  to  a  certain  proportion,  at  which  life 
was  not  manifest.  Cultures  made  from  their  atten- 


BACTERIA    INVESTIGATION.  107 

uated  microbes  retained  their  attenuation.  Ac- 
cording to  these  investigators,  the  essential  con- 
dition for  attenuating  the  virulence  of  Bacillus 
anthracis,  whether  by  the  methods  of  cultures  at 
42°-43°  C.  or  by  the  use  of  antiseptics,  is  the 
absence  of  spores  in  the  filaments  which  are  sub- 
mitted to  the  prolonged  action  of  air,  of  heat,  or 
of  diverse  chemical  agents.  The  spore  is  the  form 
of  resistance  of  Bacteridia ;  it  is  shielded  from 
the  action  of  most  environments,  and  preserves 
the  properties  of  the  filament  which  gave  it  birth. 
Notwithstanding  this  resistance  to  external  agents, 
the  germs  of  the  Bacteridia  may  be  modified  and 
attenuated  in  its  virulence  in  a  like  manner  as  the 
filament.  Some  spores  of  Bacillus  antJiracis  were 
kept  in  contact  with  sulphuric  acid  (2  :  100)  at 
35°  C.,  in  a  closed  tube,  and  frequent  shaken  in 
order  to  insure  contact  of  the  acid  and  the  spores. 
Every  two  days  a  small  quantity  was  placed  in  a 
slightly  alkaline  broth.  The  cultures  thus  obtained 
during  the  first  days  killed  rabbits  and  guinea-pigs  ; 
those  taken  at  eight  and  ten  days  killed  guinea- 
pigs,  but  were  not  fatal  to  rabbits  ;  those  taken  at 
fourteen  days  were  not  fatal  to  guinea-pigs.  In 
successive  cultures  the  germs  retained  their  attenu- 
ated virulence. 

Koch,  Gaffky  and  Loeffler's  Method  for  attenua- 
tion. They  used  the  thermostat  of  D'Arsonval 
(made  by  Wiessenegg,  in  Paris,  64  rue  Gay  Lus- 


108  THE  TECHNOLOGY   OF 

sac) ,  which  allows  only  a  tenth  of  a  degree  vari- 
ation in  temperature.  As  a  culture  vessel  they 
used  an  Erlenmeyer's  flask,  each  fiask  containing 
20  cc.  of  chicken  broth  neutralized  with  carbonate 
of  soda.  In  each  flask  they  placed  a  small  quantity 
of  virulent  anthrax  substance,  using  all  precau- 
tions against  foreign  contamination.  The  flasks 
were  then  placed  in  the  thermostat  at  42°-43°  C., 
and  the  degree  of  attenuation  ascertained  from 
time  to  time  by  small  quantities  being  taken  out 
and  inoculations  made  upon  mice,  guinea-pigs, 
rabbits,  and  sheep.  The  authors  lay  great  stress 
on  the  use  of  a  pure  culture  for  the  test  inocula- 
tions, "  contrary  to  Pasteur,  whose  vaccins  are 
often  shown  to  be  impure  with  other  Bacteria." 
Each  time  a  test  of  the  attenuation  reached  by  the 
cultures  was  made,  new  cultures  of  the  same  were 
made  in  two  new  flasks  of  sterilized  chicken  broth, 
so  that  in  at  least  one  they  should  have  a  pure 
culture.  To  fix  the  degree  of  attenuation,  the 
flasks  were  on  the  fifth  day  placed  at  37°  C., 
where  they  formed  resting  spores.  The  conclusion 
arrived  at  was  that  the  protective  inoculation 
methods  practised  up  to  the  present  time  are  of 
doubtful  advantage. 

o 

Staining.  The  Bacilli  of  anthrax  may  be 
stained  with  the  ordinary  aniline  dyes,  and  ac- 
cording to  general  directions.  They  are,  however, 
difficult  to  recognize  in  the  tissues,  if  these  are  not 


BACTERIA    INVESTIGATION.  109 

well   decolorized ;    this  is  best   done    by   Gram's 
general  method,  q.  v. 

Weiyerfs  Double- Staining  Method  (gentian- 
violet  and  picro-carmine).  Place  sections  2-5 
minutes  in  a  one  percent  aqueous  solution  of  gen- 
tian-violet. Wash  in  alcohol.  Allow  sections  to 
float  a  moment  in  water,  and  then  place  in  picro- 
carmine  until  intensely  stained  (£-1  hour).  Wash 
in  alcohol  until  color  has  faded  somewhat.  Oil  of 
cloves,  Canada  balsam. 

Feltz's  Method  of  ascertaining  the  r61e  of  earth- 
worms in  the  propagation  of  charbon  was  to 
mix  some  sterilized  earth  with  a  fluid  containing 
anthrax  Bacilli,  and  to  place  this  in  flower-pots. 
After  three  weeks  the  worms  were  removed  one 
at  a  time,  cut  up,"  after  being  thoroughly  washed, 
and  injected  into  guinea-pigs. 

LITERATURE    OF    ANTHRAX. 

ARLOING  (S.)  :  "  Influence  de  la  lumi£re  sur  la 
ve*ge*tation  et  les  proprietes  pathogenes  du  Bacil- 
lus anthracis."  Compt.  liend.  Feb.  9,  1885, 
p.  378. 

ARLOING,  CORNEVIN,  ET  THOMAS  :  I.  "  Sur 
Finoculabilite  du  charbon  symptomatique  et  les 
caracteres  qui  le  diflerencient  du  sang  du  rate." 
Compt.  Rend.  T.  90,  p.  1302  (1883).  II.  "  De 
1'inoculation  du  charbon  symptomatique  par  Pin- 
jection  intra-veineuse,  et  de  1'immunite  conferee 


110  THE   TECHNOLOGY   OF 

au  veau,  au  mouton,  eta  la  che"vre  par  ce  precede." 
Compt.  Rend.  T.  91,  p.  934. 

ARCHANGELSKI  :  "  Ueber  Milzbrand."  Ctblt. 
f.  d.  med.  Wiss.  1882,  No.  15. 

ALADER  (v.  Rozahegva)  :  T  Versuche  mit  der 
Pasteur'schen  Schutzimpfung  gegen  Milzbrand  in 
Ungarn."  Deutsche  med.  Wochenschr.,  8  Jahrg. 
1882,  p.  24. 

BERT  (P.) :  (Anthrax).  "  Compt.  Rend.  Soc- 
Biol."  for  1877.  (1879)  pp.  19,  20,  p.  317,  p.  442, 
p.  465. 

BOLLINGER:  "  Ueber  Milzbrand."  Ctblt.  f.  d. 
med.  Wissensch.,  1872. 

BRAUELL  :  Ueber  Milzbrand."  Virchow's  Ar- 
chiv.,XL,  XIV.,  XXXVI. 

BUCHNER  UND  ROBERTS  :  "  Uel)er  d.  experim. 
Erzeugung  d.  Milzbrand  contagiums,  aus  d.  Heu- 
pilzen."  Archiv.  f.  exp.  Pathpl.  u.  Pharm.-,  Bd. 
XIII.,  H.  2,  p.  170  (1880). 

BUCHNER  (H.)  :  I.  "  Ueber  Entstehung  des 
Milzbrandes  durch  Einathmung."  Sitzungsbr.  d. 
konigl.  bayr.  Acad.  d.  Wiss.,  1880.  Hft.  3 
(Mixed  Anthrax  spores  with  dust  and  allowed 
white  mice  to  breathe  it ;  in  24  cases  they  died  in 
from  1-3  days).  II.  "Ueber  die  experimentelle 
Erzeugung  des  Milzbrand  contagiums,"  ibid.  1882, 
Hft.  II. 

BOULEY  :  I.  "  De  la  vaccination  charbonneuse." 
Compt.  Rend.  T.  42,  p.  1383 ;  T.  43,  p.  190.  II. 


BACTERIA    INVESTIGATION.  Ill 

"  Sur  Pidentite  du  charbon  dans  toutes  les  especes 
d'animaux  domestique."  Compt.  Rend.  T.  84, 
p.  993  and  1877. 

BURDON-SANDERSON,  DUGUN,  GREENFIELD,  and 
BENHAM  :  "Investigations  of  Anthrax."  Journ. 
Roy.  Agric.  Soc.,  1880,  No.  31. 

CHAMBERLAND  ET  Roux :  I.,  "Sur  la  vaccina- 
tiere  charbon."  Compt.  Rend.  T.  42,  p.  1378. 
II.  "  Sur  les  germes  charbonneux,  ibid.  T.  92,  p. 
209.  III.  "De  Fattenuation  des  virus  et  leur 
retour  a  la  virulence,"  ibid.  T.  92,  p.  429.  IV. 
"La  vaccination  du  charbon,  ibid.  T.  92,  p. 
662-666.  Y.  "  Sur  1'attenuation  de  la  virulence 
de  la  bacteridie  charbonneuse,  sous  Tinfluence  des 
substances  antiseptiques,"  ibid.  T.  96,  p.  1088 
(1883).  VI.  "Sur  1'attenuation  de  la  bacteridie 
charbonneuse  et  de  ses  germes  sous  1'influence  des 
substances  antiseptiques,"  ibid.  T.  96,  p.  1410. 

CHAMBRELENT  ET  MOUSSONS  :  "  Experiences  sur 
le  passage  des  bacteridies  charbonneuses  dans  de 
lait  des  animaux  atteints  du  charbon."  Compt. 
Rend.  T.  97  (1883),  p.  1142-1145. 

CHATEAU  (A.)  :  I.  "Nouvelles  experiences  sur  la 
resistance  des  moutons  algdriens  au  sang  de  rate." 
Compt.  Rend.  T.  90,  p.  1396.  II.  "  Des  causes 
qui  peuvent  faire  varier  les  results  de  1'inoculation 
charbonneuse  sur  les  moutons  algeriens.  Influence 
de  la  quantite  des  agents  infectants,  application  a 
la  thdorie  de  Timmunite,"  ibid.  T.  90,  p.  1526, 


112  THE    TECHNOLOGY   OP 

year  1880.  III.  "  Du  renforcement  de  Fimmunite 
des  moutons  algeriens,  a  Fegard  du  sang  de  rate, 
par  les  inoculations  preventive.  Influence  de  Fin- 
oculation  de  la  mere  stir  la  receptivite  du  foetus," 
ibid.  T.  91,  p.  148.  IV.  "  Sur  la  resistance  des 
animaux  de  Fespece  bovine  au  sang  du  rate  et  sur 
la  preservation  cle  ces  animaux  par  les  inoculations 
preventives,"  ibid.  T.  91,  p.  648.  V.  "Etude 
experimental  de  Faction  exercee  sur  1'agent  infec- 
tieux,  par  1'organisme  des  moutons  ou  moins  re- 
fractaires  au  sang  de  rate  ;  se  qu'il  advient  des 
microbes  specifiques,  introduits  directement  dans 
le  torrent  circulatoire  par  transfusions  massive  de 
sang  charbonneux,"  ibid.  T.  91,  p.  680.  VI. 
"  De  Fattenuation  directe  et  rapide  des  cultures 
virulent  par  Faction  de  la  chaleur,"  ibid.  T.  96,  p. 
553  (1883).  VII.  "De  la  facultd  prolifique  des 
agents  virulent  attenues  par  la  chaleur,  et  de  la 
transmission  par  generation  de  Finfluence  attenuante 
d'un  premier  chauffage,"  ibid.  T.  96,  p.  612. 
VIII.  "  Du  r61e  de  Foxygene  de  Fair  dans  Fatte- 
nuation  quasi  instantande  des  cultures  virulent  par 
Faction  de  la  chaleur,"  ibid.  T.  96,  p.  678.  IX. 
"  Du  r61e  respectif  de  Foxygene  et  de  la  chaleur 
dans  Fattdnuation  du  virus  charbonneaux  par  la 
me'thode  de  M.  Pasteur.  Theorie  generale  de  Fat- 
tenuation  par  Fapplication  de  ces  deux  agents  au 
microbes  aerobies,"  ibid.  T.  96,  p.  1471.  X. 
"  De  la  preparation  en  grandes  masses  des  cultures 


BACTERIA    INVESTIGATION.  118 

attenuees  par  le  chauffage  rapide  pour  1'inoculation 
preventive  du  sang  de  rate,  ibid.  T.  98,  1884,  p. 
73.  XI.  "  Du  chiiuffage  des  grandes  cultures  de 
bacilles  de  sang  de  rate,  ibid.  p.  126.  XII.  "De 
1'attenuation  des  cultures  virulent  par  1'oxygene 
comprimeY'  ibid.  p.  1232. 

DAVAINE  :  "  Observations  sur  la  maladie  char- 
bonneuse."  Compt.  Rend.  (1877)  T.  84,  p.  1322. 
Comp.  also  T.  57  and  59. 1 

DUPLESSIS  :  "  Milzbrandimpfung  mit  tier  Pas- 
teur'schen  Culturlymph."  Journ.  d'Agricult.  prac- 
tique.  Bd.  II.,  S.  32. 

FELTZ  :  "  De  la  dure'e  de  Fimmunit^  vaccinale 
anticharbonneuse  chez  le  lapin."  Compt.  Rend.  T. 
98,  Aug.  4,  1884,  p.  246,  prelim,  com.  Nov.  6, 
1882.  II.  "  Sur  la  rc>le  des  vers  de  terre  dans  la 
propogation  du  churbon  et  sur  1'attenuation  du 
virus  charbonneux."  Compt.  Rend.  (1882),  T.  95, 
No.  19,  p.  859-862. 

FOKKEE  (A.  P.)  :  "  Zur  Bacteriensfrage."  Vir- 
chow's  Archiv.  Bd.  88  (1882).  II.  "  Die  Identi- 
tat  von  Bacillus  anthracis  u.  Bacillus  subtilis" 
Ctbl.  f.  d.  med.  Wiss.  1880.  No.  44. 

FRISCH  :  I.  "  Die  Milzbrandbacterien  u.  ihre 
Yegetationen  in  d.  lebendeii  Hornhaut."  Akad. 

1  Davaine  was  the  first  (1850)  to  describe  Bacillus  anthracis  in 
"  Sang  de  rate."  "  111  y  avait  en  outre  dans  le  sang  de  petits  corps 
filiform,  ayant  environ  le  double  en  longuer  des  globules  sanguin, 
ces  petits  corps  n'offraient  point  de  mouvement  spontane'." 


114  THE  TECHNOLOGY  OF 

d.  Wiss.  74.  Bd.  III.  (1876);  II.  "Ueber  das 
Yerfaalten  der  Milzbrand  bacillen  gegen  niedere 
Ternperatur."  Strieker's  med.  Jahrb.,  1879,  p. 
513. 

FEIEDLANDER  :  "  Die  Identitat  v.  Bacillus  sub- 
tilis  u.  B.  anthracis."  Ctblt.  f.  d.  med.  Wiss. 
1880.^ 

GOTTI  (A.) :  "  Sopra  alcuni  experiment!  di  in- 
oculazione  carbonchiosa  preservativa  nei  bovini." 
Mernorie  della  R.  Accademia  delle  Scienze  dell'  In- 
stitute di  Bologna.  Ser.  IV.,  T.  V.,  p.  734. 

GREENFIELD  (W.  S.) :  "On  Bacillus  anthracis." 
Proc.  Roy.  Soc.,  London,  1879-80. 

KOCH,  GAFFKY,  u.  LOEFFLER  :  "  Experimentelle 
Studien  iiber  die  kiinstliche  Abschwachung  der 
Milzbrand-bacillen  und  Milzbrand-infection  durch 
Futterung."  Mittheil.  aus  d.  kais.  Gesundheitsamt. 
Bd.  2,  1884. 

KOCH  (R.)  :  I.  "Ueber  die  Milzbrandimpfung. 
Eine  Entgegung  auf  den  von  Pasteur  in  Genf.  ge"- 
haltenen  Yortrag."  Kassel  u.  Berlin,  1882.  II. 
"  Zur  Aetiologie  d.  Milzbrandes."  Mittl.  a.  d.  kais. 
Gesundheitsamt,  1881.  III.  "Ueber  Milzbrand 
und  Milzbrandimpfung."  54  Yers.  deutsch.  Na- 
turf.  u.  Aertze.  Salzburg,  1881.  IY.  "  Milz- 
brandversuche."  Mittheilung  a.  d.  kais.  Gesund- 
heitsamt, 1881,  Berlin. 

KOUBASSOFF  :  "  Passage  des  microbes  de  la  m^re 
au  foetus."  Compt.  Rend.  T.  100  (1885),  p.  372. 


BACTERIA    INVESTIGATION.  115 

LUTZ  :  "  Eine  Milzbrand  epidemie  bei  Men- 
schen.'"  Aerztl.  Int.  BL,  1881,  21. 

OSOL  (K.)  :  "Das  Anthrax  Virus."  Ctbl.  f.  d. 
med.  Wiss.  1884,  p.  401-404. 

PASTEUR  ET  JOUBERT  :  "  Etude  sur  la  maladie 
charbonneuse."  Compt.  Rend.  T.  84,  p.  900 
(1877).  II.  "Charbon  et  septice'mie."  Compt. 
Rend.  T.  85,  p.  101 ;  also  p.  61. 

PASTEUR  (L.)  :  I.  "Sur  1'aetiologie  du  char- 
bon."  Ibid.  T.  91,  p.  86  (1880).  II.  "Experi- 
ences tendant  a  demontrer  que  les  poules  vacci- 
ne*es  pour  la  cholera  sont  refractaircs  au  charbon." 
Ibid.  T.  91,  p.  315  (1880).  III.  "Sur  1'etiologie 
des  affection  charbonneuses."  Ibid.  T.  91,  p.  455 
(1880).  IV.  "Sur  la  non  recidive  de  1'affection 
charbonneuse."  Ibid.  T.  91,  p.  531  (1880).  V. 
"Nouvelles  observations  sur  1'etiologie  et  la  pro- 
phylaxie  du  charbon."  Ibid.  T.  91,  p.  697  (1880). 
VI.  "  Announcement  that  Anthrax  germs  were 
brought  to  surface  of  ground  by  earth-worms." 
Trans,  d.  Acad.  d.  med.  a  Paris,  17  Mai, 
1881.  VII.  "  La  vaccination  charbonneuse.  Re*- 
ponse  au  Dr.  Koch."  Revue  scientifique,  Jan.  20, 
1883. 

PASTEUR,  CHAMBERLAIN,  ET  Roux :  "De  1'at- 
tenuation  des  virus  et  de  leur  retour  a  la  viru- 
lence." Compt.  Rend.  28  Feb.  1881.  II.  "Le 
vaccin  du  charbon."  Ibid.  T.  92  (1881),  p.  666. 
III.  "  Sur  la  longue  dure*e  de  la  vie  des  germes 


116  THE  TECHNOLOGY  OF 

charbonneux  et  sur  leur  conservation  dans  les 
terres  cultivees."  Ibid.  Jan.  31,  1881. 

PERRONCITO  :  "  Ueber  die  Tenaeitat  des  Milz- 
brand  Virus  in  seinen  beiden  Gestalten,  als  Spore 
u.  Bacillus  anthracis."  Revue  fur  Thierheilkunde 
u.  Thierzucht,  1883,  No.  11. 

POINCAR£  :  "  Sur  la  production  du  charbon  par 
les  parturages."  Compt.  Rend.  T.  91,  p.  179 
(1880). 

POLLENDER  :  "  Microscopische  und  chemische 
Untersuchungen  d.  Milzbrandblutes."  Gaspers 
Vierteljahrsschrift  f.  gerichtl.  Medecin.  13. 

PRAZMOWSKY  (A.)  :  "Ueber  den  genetischen 
Zusammenhang  der  Milzbrand  und  Heubakterien." 
Biol.  Centralblatt.  1884,  No.  13. 

RODET  (A.)  :  "Sur  la  rapidite  de  la  propaga- 
tion de  la  Bacteridie  charbonneuse  inoculee." 
Compt.  Rend.  10  Avril,  1882.  Journ.  de  Mi- 
crographie,  1882,  p.  408. 

ROLOFF  :  I.  "  Milzbrand  Entstehung  u.  Bekamp- 
fung."  1882.  II.  "  Ueber  die  Milzbrandimpfung 
und  entwickeln  d.  Milzbrandbacterien."  Arch.  f. 
wiss.  u.  pr.  Thierheilkunde.  Bd.  9,  Hft.  6.  p.  459. 

ROSSINGOL  (H.)  :  "Les  nouvelles  experiences 
de  Pouilly-le-Fort.  L'immuniy  conferee  par  la 
vaccination,  practiquee  avec  le  virus  charbonneuse 
attenue  de  M.  Pasteur,  est-elle  transmissible  de  la 
mere  au  foetus  ?  "  Angers  (Lechese  et  Dolbeau) , 
1883. 


BACTERIA    INVESTIGATION.  117 

STRAUSS  ET  CHAMBERLAND  :  "Passage  de  la 
bacteridie  charbonneuse  de  la  mere  au  foetus." 
Compt.  Kend.  T.  95,  No.  25  (1882). 

SZPILMAN  ( J.)  :  "  Ueber  das  Verbal  ten  der  Milz- 
brand  bacillen  in  Gasen."  Hoppe-Seyler's  Zeit- 
schrift  f.  physiol.  Chemie.  Bd.  IV.,  p.  350  (1880). 

TOUSSAINT  (H.)  :  I.  "De  1'immunit^  pour  le 
eharbon,  aquise  a  la  suite  d'inoculation  pre*ven- 
tives."  Compt.  Rend.  T.  91,  p.  135  (1880).  II. 
"De  rirnmunite  pour  la  eharbon,  aquise  a  la  suite 
des  inoculations  preventives.  Precede  pour  la  vac- 
cination du  mouton  et  du  jeurie  chien."  Ibid.  T. 
91,  p.  303  (1880). 

WEIGERT  :  (Staining  method).  Yirchow's  Ar- 
chiv.  Bd.  81. 

CHOLERA. 

(Comma  Bacillus.     Koch.) 

The  importance  of  a  knowledge  of  the  Bacteria 
inhabiting  the  healthy  body,  as  well  as  a  proper 
preliminary  training  in  the  technique  of  Bacteria 
investigation,  is  strikingly  shown  in  connection 
with  the  microbe  of  cholera.  Lewis  claimed 
that  a  Bacillus  resembling  that  of  cholera  could 
be  found  in  the  mouth ;  in  regard  to  which  Dr. 
Koch  shows,  in  a  very  few  words,  that  the  form 
referred  to  has  been  known  for  several  years, 
that  it  differs  from  the  Comma  Bacillus  in  being 
longer,  more  slender,  and  not  so  blunt  at  the 


118  THE   TECHNOLOGY   OF 

ends,  and  further  differs  in  the  important  par- 
ticular that  it  does  not  form  the  characteristic 
colonies  in  weakly  alkaline  peptone  gelatine. 
Again,  Finkler  and  Prior  claimed  to  have  found 
the  Comma  Bacillus  in  the  stools  of  cholera  nos- 
tras  patients  ;  Koch  obtained  some  of  their  culture 
material,  and  found  in  it  four  different  microbes,  of 
which  one  resembled  slightly  the  Comma  Bacillus, 
but  was  larger  and  plumper,  and  in  its  mode  of 
growth  quite  different,  growing  much  more  rapidly 
in  gelatine  or  on  potato,  and  showing  unmistakable 
differences  in  the  form  assumed  in  the  cultures. 

Koctis  Method  for  diagnosing  the  cholera  Bacil- 
lus.1 Microscopical  study  of  the  intestine  of 
cholera  patients  showed,  especially  where  the 
Peyer's  patches  were  reddened  at  the  edges,  an 
in  wandering  of  Bacteria  ;  they  were  found  partly 
in  the  glands,  partly  between  the  epithelium  and 
the  basement  membrane,  raising  up  the  epithelium. 
Some  of  the  Bacteria  had  a  peculiar  appearance  as 
to  size  and  shape,  by  which  they  could  be  distin- 
guished. The  contents  of  the  intestine  exhibited 
a  great  variety  of  Bacteria.  He  selected  two 
acute,  uncomplicated  cases,  in  which  no  blood  had 
yet  appeared  in  the  stools,  and  compared  the 
Bacilli  of  the  intestinal  contents  until  he  found 


1  Taken  from  Koch's  report  at  the  Cholera  Conference,  July 
26,  1884. 


BACTERIA    INVESTIGATION.  119 

some  similar  to  those  in  the  glands.  These  he 
named  Comma  Bacilli.  They  are  smaller  than 
the  Bacilli  of  tuberculosis,  about  two  thirds  as 
long,  but  much  plumper  and  thicker,  and  with  a 
slightly  bent  appearance,  not  usually  any  greater 
than  that  of  a  comma ;  sometimes  the  curve  is 
doubled,  so  that  an  S  shape  results.  This  arises 
from  the  two  individuals  formed  by  fission  adher- 
ing together.  In  pure  cultures  another  very  char- 
acteristic developmental  form  is  seen.  It  consists 
of  more  or  less  long,  screw-shaped  filaments,  not 
straight,  not  wavy,  but  having  a  great  resemblance 
to  the  Spirochwte  of  recurrent  fever.  Koch  thinks 
that  if  a  person  had  both  forms  together  upon  a 
slide  he  would  be  unable  to  distinguish  between 
them.  From  this  he  concludes  Comma  Bacilli  to  be 
an  intermediate  form  between  Bacillus  and  Spiro- 
chcete.  The  Comma  Bacillus  behaves  like  a  piece 
of  /Spirillum,  or  like  the  short  specimens  of  /Spiril- 
lum undula  which  do  not  make  an  entire  screw, 
but  only  short  rods  more  or  less  bent.  Cultivated 
in  meat  broths,  the  cholera  Bacilli  increase  with 
enormous  rapidity  and  in  great  abundance,  and 
are  unusually  lively.  They  grow  very  luxuriantly 
and  rapidly  also  in  milk,  but  do  not  cause  it  to 
curdle,  and  do  not  precipitate  the  casein,  as  do 
many  other  Bacteria.  The  milk  appears  unchanged, 
but  if  one  takes  a  small  drop  from  the  surface  and 
examines  it  microscopically,  it  is  found  to  teem 


120  THE  TECHNOLOGY   OF 

with  Comma  Bacilli.  They  grow  also  very  vig- 
orously in  blood  serum.  Another  good  culture 
medium  is  peptone  gelatine.  In  the  latter  they 
assume  an  entirely  characteristic  and  peculiar  form 
of  colony  formation,  such  as  Koch  had  never  seen 
elsewhere.  The  colony  appears  when  very  young 
as  an  exceedingly  pale,  small  drop,  not  perfectly 
round  in  outline  (as  is  usually  the  case  with  Bac- 
teria colonies  in  gelatine),  but  having  a  more  or 
less  irregular  border,  presenting  a  bored-out,  stel- 
late, or  ragged,  toothed  edge.  They  have  also  a 
somewhat  granular  aspect,  which  is  not  of  so 
regular  a  character  as  in  other  Bacteria  colonies. 
As  the  colony  grows,  the  granulations  become  more 
apparent,  seeming  at  length  like  a  little  heap  of 
glass  particles.  Upon  further  growth  the  gelatine 
is  dissolved  in  the  immediate  vicinity  of  the 
colony,  which  sinks  deeper  into  the  mass.  There 
is  thus  formed  a  funnel-like  pit,  in  the  middle  of 
which  the  colony  may  be  seen  as  a  small,  white 
speck.  This  behavior  is  also  peculiar  to  Comma 
Bacilli,  at  least  it  is  only  seen  in  very  few  other 
forms  of  Bacteria,  and  nowhere  so  outspoken. 
One  can  best  see  the  sinking  of  the  colony  if  he 
prepares  a  pure  culture  as  follows :  Search  out 
upon  the  gelatine  plate,  with  a  weak  power  objec- 
tive, a  suitable  colony,  pick  it  up  with  a  sterilized 
platinum  wire,  and  inoculate  a  properly  prepared 
gelatine  culture  in  a  test  tube  plugged  with  cotton. 


BACTERIA    INVESTIGATION.  121 

As  soon  as  the  culture  begins  to  develop,  one  sees 
a  small,  funnel-shaped  pit  being  formed  at  the 
point  of  inoculation.  Soon  the  small  colony  itself 
becomes  apparent,  but  remains  above  a  deep, 
sunken  spot,  which  appears  to  be  partially  fluid 
gelatine ;  there  is  also  the  appearance  as  if  an  air 
bubble  hung  over  the  colony.  It  would  seem  that 
the  vegetation  of  the  Bacilli  not  only  dissolves 
the  gelatine,  but  rapidly  thins  the  fluid  formed. 
There  are  numbers  of  other  Bacteria  which  dissolve 
the  gelatine  in  cultures,  but  in  none  other  is  there 
found  such  a  deepening,  nor  such  a  bladder-like 
cavity  on  the  surface.  The  solution  of  the  gela- 
tine gradually  progresses,  and  in  about  a  week  the 
entire  contents  of  the  tube  is  dissolved.  Special 
importance  is  placed  upon  these  peculiarities,  as 
they  serve  to  distinguish  the  Comma  Bacilli  from 
other  Bacilli.  Comma  Bacilli  may  also  be  cultivated 
in  cultures  made  with  Agar-Agar,  which  they  do 
not  dissolve  as  they  do  gelatine.  Cultures  should 
also  be  made  upon  boiled  potato,  when  they  form 
colonies  resembling  those  of  the  Bacilli  of  glan- 
ders. The  latter  form  upon  the  potato  a  thin, 
pulpy,  brown  coating.  The  colony  of  the  cholera 
Bacillus  differs  from  this  in  not  being  so  intensely 
brown,  but  rather  more  of  a  clear,  grayish-brown. 
The  Comma  Bacilli  thrive  best  at  a  temperature 
between  30°  and  40°  C.,  but  they  are  not  very 
susceptible  to  lower  temperatures ;  thus  they  will 


122  THE  TECHNOLOGY  OF 

develop  very  well,  although  somewhat  slowly,  at 
17°  C.  Below  17°  C.  the  development  is  very 
slight,  and  seems  to  cease  at  16°  C.  In  this  respect 
the  cholera  Bacilli  correspond  completely  with  the 
Bacilli  of  anthrax,  which  also  have  this  tempera- 
ture as  a  limit  to  their  growth.  Koch  made 
experiments  with  still  lower  temperatures  to  ascer- 
tain whether  these  would  not  only  stop  the  devel- 
opment, but  kill  the  microbes.  For  this  purpose 
a  culture  was  exposed  for  an  hour  to  a  tempera- 
ture of  — 10°  C. ;  the  Comma  Bacilli  were  here 
completely  frozen  for  this  length  of  time.  An 
addition  made  from  this  frozen  culture  to  fresh 
gelatine  showed  not  the  slightest  change  in  devel- 
opment; they  stood  the  freezing  well.  They, 
however,  ceased  to  grow  after  having  the  air  shut 
off  from  them.  This  can  be  shown  in  a  simple 
manner.  After  pouring  the  gelatine  culture  con- 
taining the  microbes  upon  a  glass  slide,  and  just 
before  it  stiffens,  place  upon  its  surface  a  sheet  of 
mica,  which  has  been  split  to  the  utmost  thinness 
possible,  and  which  covers  at  least  one  third  of  the 
gelatine  surface  in  the  middle.  The  mica  sheet, 
by  its  elasticity,  allows  itself  to  come  into  complete 
contact  with  the  gelatine,  and  shuts  out  the  air  from 
the  covered  spot.  It  is  now  seen,  as  the  colonies 
begin  to  develop,  that  growth  only  occurs  where  the 
gelatine  is  uncovered,  extending  under  the  edge  of 
the  mica  only  so  far  as  the  air  is  admitted,  but 


BACTERIA    INVESTIGATION.  123 

under  the  mica  itself  the  microbes  do  not  grow  at 
all.  They  here  remain  as  unusually  small  colonies, 
invisible  to  the  naked  eye,  which  have  grown  this 
much  from  using  up  the  oxygen  contained  in  the 
gelatine,  and  ceasing  to  grow  when  this  is  con- 
sumed. This  investigation  may  also  be  made  in 
another  way.  A  glass  containing  a  gelatine  culture 
medium,  which  has  been  inoculated  with  Comma 
Bacilli,  is  placed  under  the  air  pump,  which  is 
then  exhausted.  Other  glasses  prepared  in  the 
same  way  and  at  the  same  time  are  allowed  to 
remain  outside.  Those  under  the  pump  will  not 
grow.  If,  later,  these  be  restored  to  the  air,  they 
begin  at  once  to  develop.  They  have  not  been 
killed,  but  appear  able  to  increase  only  under  the 
influence  of  oxygen.  The  effect  is  similar  if  cul- 
tures are  placed  in  an  atmosphere  of  carbonic  acid, 
while  the  control  cultures  in  the  air  grow  as  usual, 
those  in  the  gas  remain  entirely  undeveloped. 
They  are  not  destroyed  by  this  treatment,  but 
begin  to  grow  if  restored  to  the  air  after  having 
been  for  a  long  time  in  carbonic  acid.  Under 
favorable  circumstances  the  Comma  Bacilli  grow 
very  rapidly.  This  vegetation  quickly  reaches  a 
maximum,  and  there  remains  stationary  for  a  short 
time,  and  then  rapidly  declines.  After  dying,  the 
Comma  Bacilli  lose  their  form,  appearing  swollen 
or  shrunken,  and  refusing  to  take  staining  alto- 
gether, or  only  slightly.  The  peculiar  vegetation 


124  THE  TECHNOLOGY  OF 

behavior  is  best  observed  by  placing  upon  damp 
earth,  substances  which,  while  rich  in  Comma 
Bacilli,  contain  at  the  same  time  other  Bacteria, 
e.  g.,  the  intestinal  contents  or  cholera  dejections 
are  placed  upon  damp  earth  or  linen,  and  kept  in 
a  damp  place.  The  cholera  microbes  here  in- 
crease in  a  most  astonishing  way  for  a  short  time. 
At  first  the  other  Bacteria  seem  to  be  stifled  by 
the  Comma  Bacilli,  which  build  for  themselves  a 
natural  pure  culture,  and  one  can  find,  by  examin- 
ing microscopically  the  surface  of  the  damp  earth, 
or  of  the  linen,  preparations  which  show  almost 
none  of  the  common  Bacteria.  They  do  not  retain 
this  luxuriant  growth  long ;  after  two  or  three 
days  they  begin  to  die,  and  the  other  Bacteria  begin 
to  increase.  This  behavior  is  exactly  similar  to 
that  in  the  intestine,  where  they  also  have  a  rapid 
increase ;  but  if  this  peculiar  vegetation  process, 
which  only  lasts  for  a  short  time,  is  over,  and 
especially  if  there  has  been  a  transudation  of  blood 
into  the  intestine,  the  Comma  Bacilli  decrease  in 
numbers,  and  the  filth  Bacteria  begin  to  develop 
more  rapidly.  From  this,  Koch  infers  that  if  the 
cholera  Bacilli  are  brought  from  the  intestine  into 
a  putrid  fluid  which  contains  much  of  the  products 
of  decomposition  of  other  Bacteria,  and  especially 
of  filth  Bacteria,  they  will  not  develop  well,  but 
will  soon  die.  Sufficient  investigation  has  not  been 
as  yet  made  upon  this  point,  which  is  important  in 


BACTERIA    INVESTIGATION.  125 

so  far  that  it  is  well  to  know  whether  the  Comma 
Bacilli  find  a  good  culture  medium  or  a  very  poor 
one  when  they  come  into  a  water-closet  or  sink. 
In  the  first  case  they  would  increase  and  require  to 
be  destroyed  by  some  disinfectant ;  in  the  latter 
case  they  would  die,  and  no  further  disinfectant  be 
needed.  Dr.  Koch  accepts  the  latter  conclusion. 

The  Comma  Bacilli  thrive  best  in  fluids  which 
do  not  contain  too  little  nourishment ;  thus,  when 
a  meat-broth  culture  was  thinned  fivefold,  it  was 
found  to  be  no  longer  useful  as  a  culture  fluid.  In 
other  experiments  they  still  continued  to  grow 
when  the  fluid  was  thinned  tenfold.  These  ex- 
periments must  evidently  be  repeated  in  a  more 
extensive  and  systematic  manner,  but  in  any  case 
it  is  known  from  the  results  obtained  that  one  dare 
not  go  too  far  in  the  dilutions,  and  that  the  Comma 
Bacilli  demand  a  certain  concentration  in  their 
nourishing  medium.  In  regard  to  cultures  it  is 
further  to  be  remarked  that  the  culture  gelatine 
and  the  meat  broths  must  not  be  of  an  acid  reac- 
tion. As  soon  as  the  slightest  trace  of  acidity 
shows  itself,  the  development  of  the  Comma 
Bacilli  is  greatly  impeded.  If  the  reaction  be- 
comes evidently  acid,  then  the  development  ceases. 
This  is,  however,  not  true  of  all  acids ;  thus,  the 
cut  surface  of  a  boiled  potato  has  an  acid  reac- 
tion from  the  presence  of  malic  acid.  Here,  how- 
ever, the  cholera  microbes  grow  luxuriantly. 


126  THE   TECHNOLOGY   OF 

There  are,  at  any  rate,  a  number  of  acids  which 
do  hinder  the  growth.  This  is  apparent  in  meat 
broth  if  lactic  acid  or  an  acid  phosphate  be  added. 

Nicati  and  Rietsctis  Method. — Pure  cultures 
of  Comma  Bacilli,  according  to  these  investigators, 
present  a  characteristic  odor  which  is  not  putrid  or 
disagreeable,  but  somewhat  ethereal  ("  e*ther£ "). 
By  means  of  a  Pasteur's  filter,  they  removed  from 
an  eight-days-old  pure  culture  in  broth  or  nutritive 
gelatine  (Koch's  formula)  all  the  Bacilli ,  and  in- 
jected the  filtrate  into  the  blood-current  of  a  dog 
(jugular  and  crural  vein),  which  had,  as  a  conse- 
quence, all  the  symptoms  of  cholera. 

Bochefontaine's  Method. — I  give  this  method, 
not  because  I  think  others  will  care  to  or  should 
repeat  it,  but  as  an  example  of  the  heroism  with 
which  investigators  carry  on  researches  for  the 
public  good,  like  impetuous  soldiers,  often  taking 
unnecessary  risks,  but  requiring  a  higher  grade  of 
fortitude  from  the  lack  of  all  excitement,  and  in 
full  knowledge  of  the  possible  results. 

Bochefontaine  made  five  large,  soft  pills  by 
incorporating  the  serous  alvine  dejections  of  a 
cholera  patient  with  gum  and  lycopodium.  These 
pills  he  took  successively,  drinking  afterwards  a 
large  glass  of  water.  Three  hours  later  the  skin 
became  hot ;  pulse  100  (66  being  in  his  case 
normal) ;  the  fever  persisted  for  twenty-four  hours, 
the  pulse  going  up  at  times  to  120  ;  slight  nausea ; 


BACTERIA    INVESTIGATION.  127 

insomnia  for  three  hours  ;  disurea  part  of  the  time  ; 
slight  convulsions  of  the  muscles  of  the  legs,  of 
the  forehead,  and  of  the  fingers  of  the  right  hand ; 
loss  of  appetite ;  and  constipation  for  twenty-four 
hours.  He  then  took  a  glass  of  purgative  alkaline 
water,  and  was  all  right  again.  The  dejections 
used  contained  microbes  of  all  kinds,  and  among 
them  the  Comma  Bacillus.  This  experiment  shows 
that  the  injection  of  the  liquid  diarrhoea  of  cholera, 
containing  Comma  Bacilli,  into  the  stomach  does 
not  necessarily  produce  cholera. 

LITERATURE. 

BIEDERT  (H.)  :  Ueber  den  Cholera-bacillus  in 
popular  wissenschaftlicher  Weise.  Kolnischen 
Zeitung,  No.  314,  1884. 

BIEDERT  (PH.)  :  ff  Die  Reinkulturen  in  Reichs- 
Gesundheitsamt  und  der  Cholera  Bacillus"  Ber- 
lin, 1885,  8vo. 

BIENSTOCK  :  (Cholera  Bacillen).  Zeitschrift  f. 
klin.  Med.  VIII.  (1884),  Hft.  1,  2. 

BOCHEFONTAINE  :  "  Experience  pour  servir  a 
1'etude  des  phenornenes  determines  chez  1'homme 
par  1'ingestion  stomacale  du  liquid  diarrheique  du 
cholera."  Compt.  Rend.  T.  99,  Nov.  17,  1884,  p. 
845. 

BORNER  (P.)  :  I.  Die  bisherige  Thatigkeit  der 
Herren  Finkler  und  Prior  zur  Aetiologie  der 
Cholera,  und  ihre  Entgegung  wider  R.  Koch  —  in 


128  THE  TECHNOLOGY  OP 

der  Kolmsche  Zeitung."  Deutsch.  med.  Wo- 
chenschr.,  No.  47,  1884,  p.  770.  II.  Weitere 
Beitrage  zu  der  Discussion  iiber  die  Cholera  Bak- 
terien."  Ibid.  No.  48,  p.  788,  Nov.  27,  1884. 

BUCHNER  (H.)  :  "Ueber  die  Koch'schen  u. 
Finkler-Prior'schen  Komma-bacillen."  Sitzungs- 
bericht  der.  Ges.  f.  Morphologic  u.  Physiol. 
in  Munchen.  Sitzung.  v.  13,  Jan.,  1885.  II. 
"  Bemerkungen  zu  Fliigge's  Kritik  der  Emme- 
rich'schen  Cholera-untersuchungen."  Sep.  Ab- 
druck  aus  d.  Berliner  klin.  Wochenschr.  1885. 
No.  15. 

EMMERICH  (R.)  :  "Ueber  die  in  choleraleichen 
und  cholerakranken  gefundenen  Pilze."  Deutsch. 
med.  Wochenschr.  1884.  No.  50 ;  also  Archiv. 
f.  Hygiene,  Jan.,  1885.  The  Lancet,  Dec.  27, 
1884. 

ERMENGEN  (E.  VAN)  :  I.  "  Contribution  a  1'^tude 
du  Microbe  du  Cholera  Asiatique.  Recherches  sur 
un  micro-organisme,  decouvert  par  MM.  Finck- 
ler  et  Prior  dans  le  cholera  sporadique,"  Journal 
de  Micrographie,  No.  XI.  (Nov.,  1884),  p.  595. 
II.  "  Recherches  sur  le  bacille  virgule  du  cholera 
asiatique,  conclusions  principales  du  travail  pr6- 
sente  a  la  Societe  Beige  de  Microscopic  dans  sa 
stance  de  26  Octobre,  1884.  Ext.  in  Deutsch.  Med. 
Wochenschr.,  No.  46,  p.  749.  Published  sepa- 
rately. Bruxelles,  1884.  pp.  37.  8vo,  avec  2 
plch.  photogr. 


BACTERIA   INVESTIGATION.  129 

FINKLER  u.  PRIOR  :  "  Untersuchungen  iiher 
Cholera  nostras."  Congress  des  Naturalists  Alle- 
mand,  a  Magdebourg,  Stance  du  20  Sept.,  1884. 
Deutsche,  med.  Wochenschr.,  No.  36  (1884), 
p.  581:  also,  No.  59,  p.  632-634.  Kolnischen 
Zeitung,  Nov.  11,  1884.  Tageblatt  der  Naturfor- 
chersers,  1884;  p.  218-223. 

KLAMANN  :  w  Bacilli  d.  Cholera  nostras."  Tage- 
blatt der  Naturforscherers,  1884,  p.  223. 

KLEBS  (E.)  :  "  Ueber  Cholera  Asiatica  "  (Cholera 
bacillen),  Basel.  1885.  (B.  Schwalbe),  pp.  18, 
gr.  8vo,  mit  Holzschnitten.  cf.  Schweizer  arztl. 
Corresp.  Bl.  1884.  No.  23. 

KLEIN  (E.) :  On  Cholera  Bacilli.  Journ.  of 
Science,  VI.,  1884,  p.  510. 

KOCH  (R.)  :  I.  Bericht  der  deutschen  wissen- 
schaftlichen  Commission  zur  Erforschung  der 
Cholera,  March  4,  1884.  Abstract  in  Fortschritte 
der  Med.  1884,  Bd.  2,  p.  68.  II.  Bericht  d. 
Conferenz  zur  Erorterung  der  Cholerafrage.  Berl. 
klin.  Wochenschr.  1884.  No.  31,  ff.  July  26, 
1884.  Abstract  in  P.  Borner's  Reichs~Med.  Kalen- 
der  f.  Deutschld.  Theil.  II.  1885.  "Ueber  die  Chol- 
era bacterieri."  Deutsche  Med.  Wochenschr.,  No. 
32  u.  32  a,  1884;  also  No.  45  (Nov.  6),  p.  725. 

LANKESTER  (E.  RAY)  :  "  On  Comma  bacilli." 
Nature,  1885,  XXXI.,  p.  168-171,  6  Figs.  cf. 
Pall  Mall  Gazette,  Oct.  6,  1884,  pp  1,  2.  Times, 
Nov.  19,  1884. 


130  THE  TECHNOLOGY   OF 

LEWIS  (T.  R.)  :  "  Memorandum  on  the  'Comma- 
shaped  Bacillus '  alleged  to  be  the  cause  of  chol- 
era." Med.  Times  and  Gazette,  Sept.  20,  1884. 
The  Lancet,  Sept.  20,  1884,  p.  513., 

MAREY  :  w  Les  eaux  contaminees  et  le  Cholera." 
Compt.  Rend.  T.  99,  Oct.  27,  1884,  p.  667. 

NICATI  UND  REITSCH  :  I. -"La  vitalite  du  Mi- 
crobe de  Cholera."  Revue  Scientifique,  No.  21, 

1884.  II.  "  Odeur  et  effets  toxique  des  produits 
de   la   fermentation   produite   par   le   bacilles   en 
virgule."     Compt.  Rend.  T.  99,  1884.     Semaine 
med.    1884.     No.    38.     Deneke    deutsche    med. 
Wochenschr.  1885.   No.  3.  Chemisches  Centralbl. 

1885.  No.  5. 

NICATI  (W.)  :  "Cholera  et  Cholemie."  Compt. 
Rend.  T.  99,  Nov.  24,  1884,  p.  929. 

PACINI  (F.) :  "  Nuove  Osservazioni  microscop- 
ische  sul  Colera."  Meniorie  inedite  racotta  e  pub- 
licate  per  cura  di  A.  Bianchi.  Milano,  1884. 

PELLETAN  (J.)  :  "Le  '  Kommabacilius '  Koch." 
Journal  de  Micrographie.  T.  8,  p.  475. 

PETTENKOFFER  (MAX  v. ) :  "  Zur  Aetiologie 
oder  Infectionkrankheiten.  1881,  pp.  333-352. 
cf.  Bot.  Centralbl.  IX.,  1882,  p.  25.  (On  the 
parasitic  nature  of  cholera.) 

POUCHET  (G.)  :  "Sur  la  presence  des  sels  bili- 
aires  dans  le  sang  des  cholerique  et  sur  1'existence 
d'un  alcaloide  toxique  dans  les  dejections."  Compt. 
Rend.  T.  98,  Nov.  17,  1884,  p.  847. 


BACTERIA  INVESTIGATION.  131 

STERNBERG  (G.  M.) :  "  The  Comma  Bacillus  of 
Koch."  Science,  Feb.  6,  1885,  p.  109. 

TAXIS  ET  CHAREYRE  :  "  La  Bacille  du  Cholera." 
Journal  de  Micrographie,  T.  VIII.,  p.  397,  444. 

UNGAR  :  "  Ueber  das  Verhalten  des  von  Finkler 
und  Prior  in  den  Stuhlentlechungen  bei  der  Cholera 
nostras  gefundenen  Bacillen."  Kolnischen  Zei- 
tung,  No.  323,  1884. 

VENTUROLI  (M.)  :  "  II  bacillo-virgola  di  Koch  e 
la  microscopia."  Bologna  (Tip.  Arcvvscovile) 
1884,  16  pp.  16rno. 

WATERS  (G.) :  "  The  Comma-shaped  Bacillus  a 
zymotogenic,  not  a  pathogenic,  entity."  Med. 
Times  and  Gazette,  1884,  Nov.  8. 

GLANDERS. 

Bacillus  malandrice  (Israel). 

Loeffler  and  Schutz  Method.  —  A  firm  culture 
medium  is  made  from  horse-blood  serum,  and 
inoculated  with  the  microbes  of  a  fresh  glander 
knot.  After  three  days  the  surface  of  the  serum 
shows  numerous  small ,  transparent  drops  ;  in  these 
are  found  fine  Bacilli,  such  as  are  found  in  the 
diseased  lung,  liver,  .spleen,  and  nasal  wall.  For 
staining,  use  a  concentrated  aqueous  solution  of 
methyl-blue  ;  treat  with  highly  diluted  acetic  acid, 
then  dehydrate  with  absolute  alcohol,  cedar  oil, 
balsam.  Inoculation  experiments  need  further 
repetition.  In  this  disease  the  Bacilli  are  very 


132  THE  TECHNOLOGY  OF 

difficult  to  find.  The  investigation  must  be  carried 
on  with  an  oil-immersion  objective,  and  an  Abbe's 
condenser.  There  is  no  peculiar  staining  method 
by  which  we  can  find  the  Bacilli  of  glanders  in 
living  animals,  since  it  is  impossible  to  stain  them 
without,  at  the  same  time,  staining  the  Bacteria 
normally  to  be  found  in  the  nasal  mucus. 

LITERATURE. 

BOUCHARD,  CAPITAN  ET  CHARVIN  :  Revue  medi- 
cale  fran9aise,  Dec.  30,  1882. 

GALTIER  (Y.) :  "  Inoculation  de  la  morve  au 
lapin ;  destruction  de  l'activit£  virulente  de  la 
morve  par  la  dedication,  transmission  de  la  morve 
par  1'inoculation  de  la  saline."  Compt.  Rend.  T. 
91,  p.  475.  II.  "Inoculation  de  la  morve  au 
chien."  Ibid.  T.  92,  p.  303. 

ISRAEL  (O.)  :  "Ueber  die  Bacillen  der  Rotz- 
krankheit"  (Vortrag,  gehalten  in  der  Sitzung  der 
Gesellschaft  der  Charitie  Aerzte  1,  Feb.  1883). 
Berliner  klin.  Wochenschrift,  1883,  No.  11,  p. 
155. 

LOEFFLER  AND  ScHUTz :  "  Ueber  den  Bacillus 
des  Rotzes."  Mittheilung  aus  dem  deutschen 
Reichsgesundheitsamt,  Deutsche  med.  Wochen- 
schrift, No.  51,  1882.  ..Berliner  klin.  Wochen- 
schrift, Jan.  1883,  p.  27. 

STRUCK:  Deutsche  med.  Wochenschr.  Nos.  51, 
52,  Dec.,  1883. 


BACTERIA    INVESTIGATION.  133 

YULPIAN  ET  BOULEY  :  "  Sur  une  note  communi- 
quee  a  1'Academie  sur  la  culture  du  microbe  de  la 
morve  et  sur  la  transmission  de  la  maladie  a  1'aide 
des  liquides  de  culture,  par  MM.  Bouchard  Capi- 
tan  et  Charrin."  Bull,  de  1'Acad  de  Medicine, 
1883.  N.  41,  Seance  du  30  Oct. 

"  The  Bacillus  of  Glanders,  New  Mounting  Me- 
dium," The  Microscope.  Vol.  IV.,  1884,  No.  4, 
p.  79. 

WASSILIEFF  :  "  Die  Bacillen  des  Rotzes  und 
ihre  Bedeutung  fiir  die  Diagnose."  Deutsche 
med.  Wochenschrift,  1883,  No.  11. 

HOG   CHOLERA. 

"  Pneumoenteritis  suis   (bacillaris)."     Bacillus  minimus 
(Klein).  —  "  Swine  plague  Schizophyte."     Detmers. 

As  in  so  many  other  cases  there  is  a  difference 
of  opinion  as  to  whether  the  pathogenic  microbe 
of  hog  cholera  is  a  Bacillus ,  or  a  dumb-bell  Mi- 
crococcus  (Diplococcus).  Klein  still  maintains  the 
correctness  of  his  conclusions,  and  claims  that 
Pasteur  is  in  error  in  all  his  experiments  which 
point  to  its  being  a  Micrococcus.  Klein  takes  no 
notice  whatsoever  of  the  investigations  of  Detmer's, 
of  which  Pasteur  speaks  as  follows  :  — 

?<  La  verite  historique  toute  fois  m'oblige  a  d6- 
clarer  qu  'en  1882,  et  egalement  au  mois  de  mars, 
le  microbe  du  rouget  avait  ete  signale  a  Chicago, 
en  Amerique,  par  le  professeur  Detmers,  dans  un 


134  THE  TECHNOLOGY   OP 

travail   qui   fait    grand    honneur   a    son   auteur." 
Compt.  Kend.  T.  97,  p.  1164. 

LITERATURE. 

DETMERS  (H.)  :  "  Remarks  on  a  pathogenic 
Schizophyte."  Micrococci  of  swine  plague  or  hog 
cholera.  Ann.  and  Mag.  of  Nat.  Hist.  5th  ser.  vol. 
VII.,  p.  471.  Extract  from  '  Science,'  May  7,  1881, 
read  before  State  Microscopical  Society  of  Illi- 
nois, April  8,  1881.  —  Amer.  Naturalist,  March, 
1882.  — Journal  de  Micrographie,  1882,  vol.  VI., 
pp.  172,  223,  496. 

EGGELING  :  "  Ueber  den  Rothlauf  der  Schweine." 
Fortschritte  der  Medicin,  Bd.  1,  Ar.  23,  p.  793, 
1883. 

KLEIN  :  "  Report  on  Infectious  Pneumoenteritis 
of  the  Pig."  Report  of  the  Med.  Office  of  the 
Privy  Council,  1877-78:  London.  "Die  Bac- 
terien  der  Schweinseuche."  Virchow's  Archiv,  Bd. 
79,  p.  468. 

PASTEUR  :  (Microbe  d.  Schweinrocken)  Mittheil- 
ungen  d.  Akad.  d.  Med.,  Nov.  27,  1883. 

PASTEUR  ET  THUILLIER  :  "La  vaccination  du 
rouget  des  pores  a  Faide  du  virus  mortal  attenue 
de  cette  maladie."  Compt.  Rend.  T.  97,  p.  1163. 

SALMON  :  (On  swine  plague).  Rep't  Dep't  Ag- 
ricult.  Washington,  1880-81 ;  see  also  article  on 
"  Contagious  diseases  of  domestic  animals."  Ibid. 
for  1884. 


BACTEEIA    INVESTIGATION.  135 


HYDROPHOBIA. 
Bacillus  lyssce  (Pasteur). 

There  is  still  room  for  careful  research  in  regard 
to  the  specific  microbe  of  hydrophobia,  opinions 
differing  as  to  whether  it  is  a  Bacillus  or  a  Micro- 
coccus.  According  to  Pasteur  it  is  the  former; 
according  to  Gibier  and  Rabe  the  latter. 

Gibier' s  Method  of  Attenuation .  —  The  virus  is, 
according  to  this  plan,  deprived  of  a  portion  of  its 
virulence  by  exposure  to  very  low  temperatures. 
It  was  found  that  no  change  was  produced  by  cold 
from  0°  to  35°  C.,  but  when  the  virus  was  kept  for 
eight  hours  at  a  temperature  of  35°  C.,  the  animals 
did  not  all  die ;  at  40°  to  43°  C.  the  dogs  and 
rabbits  resisted  its  effects,  and  were  only  rendered 
slightly  sick.  Gibier  did  not  prove  whether  this 
inoculation  gave  an  immunity  to  hydrophobia. 
His  method  of  inoculation  was  to  make  a  hole,  by 
means  of  a  small  drill,  in  the  median  line  of  the 
skull ;  the  virus  being  injected  by  means  of  a  hypo- 
dermic syringe  passed  through  the  opening  thus 
made.  The  dogs  are  kept  quiet  by  means  of  a 
hypodermic  injection  of  morphia  at  the  base  of  the 
ear. 

Pasteur's  Method  of  Inoculation.  —  Pasteur  tre- 
panned the  animal,  and  injected  the  virus,  mixed 
with  water,  upon  the  brain  by  means  of  a  Pravaz 
syringe.  After  a  period  of  from  fifteen  to  twenty 


136  THE  TECHNOLOGY   OF 

days,  the  animals  died  of  hydrophobia,  and  a  bit 
of  their  brain  was  always  capable  of  producing 
hydrophobia  in  other  animals. 

M.  Pasteur  sums  up  the  results  of  his  experi- 
ments as  follows  :  — 

I.  If  the  virus  of  rabies  is  passed  from  a  dog 
to  a  monkey,  and  then  from  one  to  other  monkeys, 
it  gradually  becomes  weaker.     If  it  is  then  injected 
into  a  dog,  rabbit,  or  guinea-pig,  it  remains  in  the 
attenuated  condition. 

II.  The   virulence  of   the    poison    is    increased 
when  it  is  passed  from  rabbit  to  rabbit,  or  from 
guinea-pig   to    guinea-pig.     If  in   this  "  exalted " 
condition  it  is  passed  into  a  dog,  it  gives  a  rabies 
which  is  always  mortal  in  effect. 

III.  Although  one  can  thus  increase  the  viru- 
lence   of  the    poison   by  passing   it    from  one  to 
another  rabbit,  it  is  necessary  to  do  several  times 
if  one  is  making  use  of  a  virus  which  has  been 
attenuated  by  a  monkey. 

Thanks  to  these  observations,  Pasteur  has  been 
able  to  preserve  an  organism  from  the  effects  of 
more  active  virus,  by  the  use  of  that  which  is  less 
so.  Here  is  an  example :  —  Virus  made  more 
powerful  by  passage  through  several  rabbits  is 
inoculated  into  a  dog,  but  it  is  inoculated  into  the 
dog  at  every  stage  of  the  experiments  on  rabbits ; 
the  result  is,  that  the  dog  becomes  entirely  refrac- 
tory to  the  most  virulent  virus." 


BACTERIA    INVESTIGATION.  137 

"  line  des  plus  grandes  difficulty's  des  recherches 
sur  la  rage  consiste,  d'une  part,  dans  1'incertitude 
du  deVeloppement  du  mal  a  la  suite  des  inocula- 
tions ou  des  morsures,  et  d'autre  part  dans  la 
duree  de  1'incubation,  c'est-a-dire  dans  le  temps  qui 
s'ecoule  entre  1'introduction  du  virus  et  1'apparition 
des  sympt6mes  rabique.  C'est  un  supplice  pour 
I'experimentateur  d'etre  condamne  a  attendre,  pen- 
dant des  mois  entiers,  le  resultat  d'une  experience, 
quand  le  sujet  en  comport  de  tr£s  nombreuses. 
On  apprendra  done,  je  1'espere  avec  un  vif  interest, 
que  nous  sommes  arrives  a  diminuer  considerable- 
ment  la  duree  d'incubation  de  la  rage  et  a  la  com- 
muniquer  a  coup  sur.  On  arrive  a  ce  double 
re"sultat  par  1'inoculation  directe  a  la  surface  du 
cerveau,  en  ayant  recours  a  la  trepanation  et  en  se 
servant  comme  matiere  inoculante  de  la  substance 
cdrdbrale  d'un  chien  enrage,  prelevee  et  inoculee 
a  1'etat  de  purete.  Pasteur,  Cornpt.  Kend.  T.  92, 
p.  1260. 

Babe's  Staining  Method,  for  making  cover-glass 
preparations  of  saliva,  was  by  the  use  of  methyl- 
violet,  according  to  general  rules.  Fuchsin  solu- 
tions, made  alkaline  with  aniline  oil,  may  also  be 
used. 

In  what  manner  the  specific  microbes  are  to  be 
distinguished  from  those  normally  existing  in  the 
saliva,  we  are  not  told  in  any  of  these  methods, 
and  there  appears  to  have  been  no  such  isolation. 


138  THE  TECHNOLOGY   OF 

Some  of  Koch's  remarks  in  reference  to  Pasteur's 
methods  would  therefore  seem  to  be  justified  in 
this  instance ;  for  example,  the  following  (taken 
from  Koch's  article  "  Ueber  die  Milzbrandimpfung  : 
Eine  Entgegung  auf  den  von  Pasteur  in  Genf. 
gehaltenen  Vortrag,"  Berlin  1882,  p.  5,  6,  7)  : 
"Pasteur  takes,  in  the  first  place,  the  position 
that  all  infectious  diseases  are  parasitic,  produced 
by  microbes,  and  he  appears  to  regard  the  con- 
ditions advanced  by  me  for  identifying  the  mi- 
cro-organism and  investigating  its  presence  in 
the  body  as  unnecessary.  For  example,  Pasteur 
makes  no  declaration  whether  he,  in  the  inves- 
tigation of  the  disease  called  by  him  nouvelle 
maladie  de  la  rage,  examined  microscopically, 
above  all  things,  the  sub-lingual  gland,  for  the 
presence  of  specific  microbes.  In  this  case  such 
an  investigation  is  indispensable,  for  it  is  known 
that  in  rabies  the  sub-lingual  glands  contain  the 
infectious  material,  and  since  the  tissues  of  the 
sub-lingual  glands  are  not  ordinarily  the  seat  of 
Bacteria,  the  supposed  specific  microbe  may  most 
certainly  be  found  here.  Pasteur,  however,  in 
conveying  the  disease  from  a  child,  dead  of  rabies, 
to  an  animal,  used  as  a  vaccine,  not  the  tissue  of 
the  sub-lingual  gland,  but  the  saliva  itself,  in 
which  it  is  known  an  immense  number  of  bacterial 
forms  are  to  be  found,  pathogenic  forms  in  healthy 
men.  The  microbes  of  hydrophobia,  which  they 


BACTERIA    INVESTIGATION.  139 

hoped  to  find  at  that  time,  and  which  are  still 
sought  for  in  vain,  Pasteur  did  not  find,  but  in- 
stead, Bacteria  were  found  which  were  considered 
as  the  cause  of  a  new  disease.  Upon  examining 
more  closely  this  'new  disease,'  it  was  soon  found 
to  be  the  already  long  known  septicaemia  of  rab- 
bits." 

The  very  fact  that  Pasteur  laid  himself  open  to 
such  criticism  as  the  above  shows  how  extremely 
careful  the  investigator  must  be,  to  leave  no  method 
untried  before  announcing  to  the  world  a  new  dis- 
covery connected  with  pathogenic  Bacteria. 

LITERATURE. 

CHAMBERLAND  ET  Roux  :  "  L'organisme  micro- 
scopique  trouve  dans  la  maladie  nouvelle  provoque 
par  la  saliva  d'un  enfant  mort  de  la  rage."  Journ. 
de  Pharm.  et  Ch6m.  T.  3. 

GIBIER  (P.):  I.  "  Becherches  sur  la  rage" 
Compt.  Rend.  T.  98.  1884.  p.  55.  II.  Recherches 
experimental s  sur  la  ,rage.  1°.  les  ouiseaux  con- 
tractent  la  rage.  2°.  ils  guerissent  spontanement 
Ibid.  T.  98,  p.  531.  Feb.  25,  1884.  III. 
"Recherches  sur  le  rage."  Ibid.  T.  96,  p.  1701 
(1883). 

LINDEMANN  :  "  Zur  Pathogenese  der  Lyssa 
humana."  Berl.  klin.  Wochenschr.  1879.  No.  4. 

PASTEUR,  CHAMBERLAND  AND  Roux  :  "  Nouvelle 
Communication  sur  la  rage."  Compt.  Rend.  T. 


140  THE  TECHNOLOGY   OF 

98,  No.  8,  p.  477.  (1884)  Ibid.  No.  20,  p. 
1229.  II.  "Le  virus  de  la  rage  atte^nueY'  Jour- 
nal dc  Micrographie,  T.  8,  p.  345. 

PASTEUR,  CHAMBERLAND,  Roux,  ET  THUILLER  : 
"  Nouvelles  faits  pour  servir  a  la  Connaissance  de 
la  rage."  Ibid.  1882.  T.  95,  No.  24.  "Sur 
la  rage."  Ibid.  T.  92  (1881),  p.  1259. 

PERCHERON  (G.)  :  "  La  rage  et  les  experiences 
de  M.  Pasteur."  Paris  (Firmin  Didot),  1884, 
149  pp.,  18mo. 

RABE  :  Revue  fur  Thierheilkunde.  No.  1. 
Bd,  VII. 

LEPROSY. 

Bacillus  leprce  (Hansen). 

The  staining  of  this  microbe  in  cover-glass 
preparations  is  only  possible  when  done  immedi- 
ately after  drying.  According  to  Baumgarten,  the 
staining  is  possible  after  a  length  of  time,  provided 
the  strength  of  the  solution  (fuchsin)  be  dimin- 
ished. If  too  great  a  time  has  elapsed,  the  Bacilli 
will  take  no  staining.  The  lepra  Bacilli  are  the 
only  yet  known  organisms  which  possess  the  pecu- 
liarity of  tubercle  Bacilli  of  not  giving  up  in 
strongly  acid  solutions  the  stain  which  they  have 
previously  taken  on.  Like  the  Bacilli  of  typhoid 
fever,  the  microbe  of  leprosy  shows  small  un- 
stained portions.  Gentian-violet,  methyl-blue, 
methyl-violet,  and  fuchsin,  stain  Bacillus  leprce; 
Bismarck- brown  does  not. 


BACTERIA    INVESTIGATION.  141 

Babe's  Method  for  Staining.  —  Prepare  accord- 
ing to  general  directions,  and  stain  in  a  solution 
of  rosanilin-chlorhydrate  in  aniline  water  (F.  36). 
Decolorize  in  a  nitric  acid  solution  (33£  per 
cent) .  Stain  the  nuclei  present  in  the  preparation 
with  methyl-blue.  A  better  method,  showing 
the  difference  of  taking  and  losing  the  stain  of 
lepra  and  tubercle  Bacilli,  is,  — 

Baumgarten's  Differentiation  Method.  —  1.  For 
Cover- Glass  Preparations,  add  five  drops  of  a  satu- 
rated alcoholic  solution  of  fuchsin  to  a  watch-glass 
of  distilled  water.  Upon  this  mixture  allow  freshly 
prepared  cover-glasses  to  float  for  six  or  seven 
minutes.  Decolorize  for  fifteen  seconds  in  acidu- 
lated alcohol.  (Nitric  acid  1  part,  alcohol  10 
parts).  Restain  with  an  aqueous  solution  of 
methyl-blue,  wash  in  water,  pass  through  absolute 
alcohol,  oil  of  bergamot,  and  mount  in  balsam. 
Study  with  a  one-twelfth  oil  immersion  objective 
with  open  condenser.  Tubercle  Bacilli  do  not  stain 
by  this  method! 

2.  For  Sections,  allow  the  sections  to  remain 
twelve  or  fifteen  minutes  in  above  staining  fluid, 
decolorize  for  thirty  seconds  in  acid  solution,  wash 
three  or  four  minutes  in  water,  absolute  alcohol, 
etc.,  or  allow  the  sections  to  remain  for  two  or 
three  minutes  in  fuchsin-aniline  water  (F.  17.), 
decolorize  for  thirty  seconds  in  above  acid  mix- 
ture. Restain  with  a  concentrated  aqueous  solu- 


142  THE   TECHNOLOGY   OF 

tion.  of  methyl-blue  for  two  or  three  minutes ; 
absolute  alcohol,  oil  of  bergamot,  Canada  balsam. 
(Tubercle  Bacilli  do  not  stain  by  this  method! 

3.  "Cover-glass"  and  dry  preparations  are  to 
be"  made  according  to  Baumgarten's  method  for 
tubercle  Bacilli  (q.  v.)  (although  the  above  some- 
what dilute  staining  fluid  may  be  used) .  Lepra 
Bacilli  assume,  after  two  or  three  minutes,  a  red 
color,  while  those  of  tuberculosis  appear  entirely 
colorless. 

JVeisser's  Methods.  — Neisser  remarks,  in  regard 
to  staining  (1.  c.  p.  524).  In  preparations  har- 
dened in  alcohol  and  not  stained,  the  Bacilli  are 
invisible.  Strong  acetic  acid  renders  them  some- 
what visible.  Caustic  potash  (1  :  12)  gives  better 
results.  They  are  well  stained  in  gentian-violet 
or  methyl- violet,  and  better  in  fuchsin.  At 
best,  they  are  difficult  to  stain,  and  the  best  results 
are  to  be  obtained  in  specimens  handled  in  weak 
caustic  potash  solution,  then  stained  and  washed 
in  acidulated  alcohol.  The  Bacilli  in  the  gentian- 
violet  preparations  are  to  be  distinguished  by  their 
red  tone  from  the  nuclei,  which  are  blue.  Dahlia 
gives  in  acid  solution  useful  preparations.  Methyl- 
blue  is  useful  only  in  showing  the  formation  of 
vacuoles  in  the  old  lepra  cells.  A  preparation 
of  eosin  and  hsematoxylin  (F.  14.)  is  useful. 
The  sections  after  staining  are  to  be  washed  in 
water  and  decolorized  in  alcohol.  The  nuclei 


BACTERIA    INVESTIGATION.  143 

show  themselves  stained  a  beautiful  blue,  the  ordi- 
nary cell  protoplasm  eosin-rose,  and  the  protoplasm 
of  cells  containing  Bacilli  a  light  orange,  so  that 
the  Bacilli  can  be  found  with  weak  powers.  Some 
of  his  preparations  lost  their  color  after  twenty- 
four  hours,  some  retained  it  for  two  years,  differ- 
ences which  he  cannot  explain. 

Neisser's  Culture  Methods  consisted  of  remov- 
ing a  lepra  tubercle  which  was  still  covered  with 
sound  skin  after  having  thoroughly  cleaned  the 
latter,  employing  in  the  process  of  extirpation  the 
greatest  precautions  against  foreign  infection. 
Cultures  were  then  made  from  this,  in  closed 
slides  with  blood-serum,  or  in  sterilized  test  tubes 
with  blood  serum,  or  with  alkaline  meat-extract 
solution.  All  preparations  were  kept  in  an  oven 
at  35°-39°  C. 

LITERATURE. 

ARNING  (E.)  :  "Ueber  das  Yorkommen  des  Ba- 
cillus leprse  bei  Lepra  ansesthetica  sive  nervosum." 
Virchow's  Archiv,  T.  90,  p.  170. 

BABES  (V.) :  (Budapest)  "  Etude  comparative  des 
bacte'ries  de  la  lepra  et  de  latuberculose."  Compt. 
Eend.  de  Tacad  de  Sc.  17  Sept.,  1883.  II. 
"  Note  sur  le  rapport  des  bacilles  de  la  tuberculose 
et  de  la  lepre  avec  les  surfaces  tegmenteuses." 
Compt.  Rend,  des  Sci.  de  la  Soc.  de  Biol,  1883, 
avril  21.  III.  "  Observations  sur  la  topographic 


144  THE  TECHNOLOGY  OP 

des  bacilles  de  la  l£pre  dans  les  tissus  et  sur  les 
bacilles  du  cholera  des  poules."  Archiv.  de  phys. 
normal  et  path.  1883.  No.  5. 

BAUMGARTEN  :  "  Ueber  Untersuchungsmethoden 
zur  Unterscheidung  von  Lepra-  und.  Tuberkel- 
Bacillen."  Zeitschrift  fiir  wissenschaftliche  Mi- 
kroskopie.  Yon  Dr.  Behren's.  Bd.  1.  H.  3.  p. 
307. 

CORNIL  ( Y. )  :  "  Note  sur  la  siege  des  bact£ries 
dans  la  lepre  et  sur  les  lesions  des  organes  dans  cette 
maladie."  Bull  de  1'acad.  d.  Med.  No.  9,  also  in 
Gaz.  Med.  d.  Paris.  No.  44,  and  Union  Med. 
No.  178-179. 

DAMSCH  :  "  Uebertragungsversuche  von  Lepra 
auf  Thiere."  Virchow's  Archiv.  Bd.  92  Heft.  1. 

GAUCHER  (E)  :  "  Culture  des  bacte'ries  de  la 
le*pra."  Gaz.  med.  de  Paris,  No.  25. 

GAUCHER  UND  HALLAIRET  :  "  Untersuchen  iiber 
den  Lepra  Parasiten."  Soc.  de  Biologie,  11  Dec., 
1880.  Kevue  de  Med.  1881,  p.  71. 

HANSEN  (AM.):  "  Bacillus  Leproe"  Yirchow's 
Archiv.  Bd.  79,  also  Quarterly  Journal  of  Mi- 
croscop.  Sci.  Jan'y,  1880.  II.  "  Studien  iiber 
Bacillus  Leprse."  Yirchow's  Archiv,  T.  90,  p.  542. 

KOBNER  (H.)  :  "Uebertragungsversuche  von 
Lepra  auf  Thiere."  Yirchow's  Archiv.  Bd.  88. 
p.  282. 

KOCH  (R.) :  Mittheilungen  aus  den  Eeichs. 
Gesundheitsamt.  p.  10.  1881. 


BACTERIA    INVESTIGATION.  145 

MEISSEN:  "  Zur  Aetiologie  der  Lepra."  Vir- 
chow's  Archiv.  Bd.  84. 

MORETTI  (O.)  :  "II  primo  caso  di  lebbra  nelle 
Marche  confermato  dalla  presenza  del  Bacillus 
leprce."  8°.  10.  pp.  con.  4  taf.  (Bologna)  Milano 
(L.  Vallardi)  1883. 

MULLER  (F.)  :  "Ein  Fall  von  Lepra."  Deutsches 
Archiv.  f.  klin.  Med.  34,  p.  205. 

NEISSER  (Dr.  Albert)  :  "  Weitere  Beitrage  zur 
Aetiologie  der  Lepra."  Virchow's  Archiv.  Bd.  84. 
1881.  p.  514-543.  Taf.  XII.,  Also  in  Breslauer 
Aerzt'l.  Zeitschr.  1879.  No.  20-21. 

THIN  :  "  Reports  on  leprous  infiltration  of  the 
epiglottis,  and  its  dependence  upon  the  Bacillus 
leprce."  Brit.  Med.  Journ.  1884.  July  19. 

x 

MALARIA. 

Bacillus  malarice  (Klebs). 

Klebs  and  Tommassi-CrudelWs  Method.  In- 
fectious material  was  obtained  from  the  air  of 
malarial  districts  by  the  use  of  a  fan  ventilator 
worked  by  hand.  This  instrument  brought  large 
volumes  of  air  with  great  force  against  a  glass 
plate  standing  at  right  angles  to  the  air  current ; 
this  plate  was  covered  with  glycerine  gelatine,  in 
which  the  particles  contained  in  the  air  fixed  them- 
selves (the  germs  from  stagnant  water  did  not 
seem  to  be  the  carriers  of  malaria).  From  the 
material  obtained  from  the  air,  fractional  cul- 


146  THE  TECHNOLOGY  OF 

tures  were  made  in  fluid  media  of  various  kinds. 
Some  of  these  pure  cultures  were  then  filtered 
through  gypsum  and  other  filters,  and  inoculation 
experiments  made.  Animals  inoculated  with  the 
unfiltered  cultures  were,  as  a  consequence,  sick 
with  typical  malarial  fever,  while  those  inoculated 
with  the  filtrate  showed  only  a  slight  rise  of  tem- 
perature, and  no  periodical  rise. 

The  various  forms  were  studied  microscopically  in 
air  chambers  coated  with  white  of  egg,  and  by  stain- 
ing with  anilin- violet,  according  to  general  rules. 

For  the  study  of  earth,  they  placed  upon  a  sand- 
bath  a  porcelain  vessel  having  a  large  surface  ;  this 
was  filled  with  the  damp  earth  in  layer  of  about 
5  cm.  thick,  and  was  kept  moist  by  the  addition, 
from  time  to  time,  of  a  little  water.  Upon  this 
prepared  earth  was  placed  a  metal  box  containing 
the  earth  to  be  examined ;  the  bottom  of  this  box 
was  pierced  with  numerous  small  openings.  The 
whole  apparatus  was  then  kept  at  a  temperature 
of  30°-35°  C.  Many  of  the  organisms  present  in 
the  earth  (diatoms,  desmids,  Hypomycetes,  etc.) 
died,  and  were  eliminated  without  any  appearance 
of  putrefaction.  Fractional  cultures  were  then 
made  from  the  earth  contained  in  the  box,  and  ani- 
mals inoculated  from  the  pure  cultures.  Clay  cyl- 
inders or  plaster  of  Paris  cylinders  were  used  as 
filters,  but  it  was  found  that  Swedish  filter-paper 
would  also  keep  back  the  Bacillus  malarim. 


BACTERIA    INVESTIGATION.  147 

Richard's  Method.  Richard  followed  in  part 
the  procedure  of  Laveran,  which  consisted  of 
direct  examination,  without  any  additional  fluid, 
of  the  blood  taken  from  a  patient's  finger  by  prick- 
ing. It  is  necessary  to  take  a  very  small  drop  of 
blood  in  order  to  have  the  blood  corpuscles  well 
separated  one  from  another.  This  method  is  the 
only  one  to  be  employed  for  studying  the  microbe 
and  its  movements ;  but  it  is  insufficient,  as  it  does 
not  disclose  the  parasitic  corpuscles  in  blood  which 
contains  but  few.  To  bring  them  out  under  these 
circumstances  he  proceeds  to  destroy  the  normal 
red  corpuscles,  by  mixing  with  the  drop  of  blood 
a  drop  of  acetic  acid.  The  parasites  are  not  de- 
stroyed, but  are  found  with  great  facility.  This 
method  has  the  advantage  that  it  preserves  for 
some  time  the  elongated  parasitic  corpuscles,  and 
them  only.  This  microbe  has  a  special  habitat, 
according  to  Richard,  the  red  blood-corpuscles  in 
which  it  develops  itself,  "  comme  un  charan^on 
dans  un  lentil le  et  d'ou  il  sort  une  fois  qu'il  est 
arrive  a  1'etat  parfait." 

LITERATURE. 

BOCHMANN  :  "  Zur  Lehre  von  der  Febris  inter- 
mittens,"  Yorlaufige  Mittheilung,  Centralbl.  f.  d. 
med.  Wissensch.  No.  33,  1880. 

CECI  (A.):  "Del  germi  ed  organism!  inferior! 
contenuti  nelle  terre  malariche  e  commune,"  4°  118 


148  THE  TECHNOLOGY  OF 

pp.  (Roma),  Milano  (L.  Vallardi),  1883.  II. 
"L'action  de  la  Quinine  (hydrochlorate)  en  rapport 
avec  le  developpment  du  germs  et  des  organismes 
inferieurs  qui  trouvent  dans  les  terres  malariques 
(Malaria)."  Trans.  Internat.  Med.  Congress  (Lon- 
don). Materia  Medica,  Rome,  1882. 

CUBONI  AND  MARCHIAFAVA  :  "  Neuen  studien 
iiber  die  Natur  der  Malaria"  ("  Nuovi  studj  sulla 
natura  della  malaria,"  Roma,  1881),  Archiv.  f. 
experim.  Pathol.,Bd.  XIII.,  p.  265. 

KLEBS  (EDWIN)  and  TOMMASI-CRUDELI  :  I. 
"Sulla  natura  dell'  agente  specitico  cbe  produce 
le  febbri  da  malaria,"  Reale  Academia  dei  Lincei, 
Vol.  III.,  Ser.  3,  del  1  gingno,  1879,  also  in 
Archiv.  f.  experim.  Pathol.  u.  Pharm.,  Bd.  XI., 
p.  126  (chap.  II.  on  methods  of  investigation) ;  II. 
"Einige  Satze  iiber  die  Ursachen  der  Wechsel- 
fieber  und  die  Natur  der  Malaria,"  Ibid.  Bd.  XI., 
Heft  2,  p.  122,  also  Heft  5  and  6,  p.  311 ;  Tab. 
II.— VI.,  III.,  "Studien  iiber  die  Ursache  des 
Wechselfiebers  und  der  Malaria."  Ibid.  Bd.  XII. 

LANZI  AND  TERRIGI  (attribute  the  cause  of  mala- 
rial fever  to  a  Bacterium  which  appears  inside  of  a 
green  alga)  :  Centralbl.  f.  med.  Wiss.,  1876, 
No.  40. 

LAVERAN  (A.)  :  "De  la  nature  parasitaire  des 
accidents  de  Timpaludisme."  Compt.  Rend.  T.  93 
(1881),  p.  627-30.  Rev.  Intern.  Sci.,IV.  (1881), 
p.  459-61. 


BACTERIA    INVESTIGATION.  149 

MARCH  AND  :  "  Kurze  Bemerkung  zur  Aetiologie 
der  Malaria,"  Virchow's  Archiv.,  Bd.  88,  p.  104. 

RICHARD  :  I.  "  Bestatigung  der  Laveran'schen 
Beob.  iiber  d.  Malaria-parasiten,"  Compt.  Rend. 
No.  8,  1882  ;  II.  "Parasiten  der  Malaria,"  Ref.  in 
Archiv.  der  Pharmacie  220,  p.  463. 

ROZSAHEGGI  (A.)  :  "Yon  der  Ursache  des 
Wechselfiebers,"  Biol.  Centralbl.,  Bd.  II.,  1881, 
p.  97. 

SEHLEN  :  "  Studien  iiber  Malaria."  Fortschritte 
der  Medicin.  1884.  No.  18. 

STERNBERG  (G.  M.)  :  "On  the  Aetiologie  of 
Malarial  Fever."  National  Board  of  Health  Bul- 
letin, Supplement,  No.  14,  Washington,  23  July, 

1881,  4  pi. 

TOMASSI-CRUDELI  (CONRAD)  :  I.  "  Der  Bacillus 
Malarice  im  Erdboden  von  Selinunte  und  Campo- 
bello,"  Archiv.  f.  experim.  Pathol.  u.  Pharm., 
Bd.  XII.,  H.  3,  p.  225;  II.  "Die  Malaria  von 
Rom,"  Deutsch  von  Dr.  A.  Schaste,  M.  Rieger, 
Munchen,  1882  ;  III.  "Die  Malaria  von  Rom  und 
die  alte  Drainage  der  romischen  Hiigel,"  Munchen, 

1882,  30  pp. 

ZIEHL  :  "  Einige  Beobachtungen  iiber  den  Bacil- 
lus Malarioe  (Klebs)."  Deutsche  med.  Wochen- 
schr.,  No.  48,  1882. 


150  THE  TECHNOLOGY  OF 

MALIGNANT  (EDEMA. 

Bacillus  oedema  (Koch). 

This  is  the  same  form  which  Pasteur  names 
:?  Vibrione  septique"  or  "  Vibrione  pyogenique" 
the  latter  being  the  microbe  of  puerperal  fever. 

Heiberg  and  Laffler  associate  Micrococci  with 
puerperal  fever.  There  is  evidently  room  for  a 
series  of  careful  experiments  in  connection  with 
this  disease. 

Malignant  oedema  is  also  known  as  "Pasteur's 
septicgemia,"  and  is  distinct  from  "  Davaine's  septi- 
caemia," which  is  caused  by  a  Bacterium. 

According  to  JFlugge,  the  Bacilli  of  malignant 
oedema  have,  when  stained  with  the  aniline  dyes,  a 
somewhat  characteristic  granular  appearance. 

Pasteur's  Method  in  puerperal  fever  was  to  cul- 
tivate blood  taken  from  the  finger  of  the  patient  in 
chicken  broth  (culture  experiments  with  the  fetid 
lochia  were  without  result).  Rabbits  and  sheep, 
into  the  abdomen  of  which  five  drops  of  the  cul- 
tures had  been  introduced,  developed  within  forty- 
eight  hours  immense  abscesses,  which  opened 
spontaneously.  Cultures  made  from  the  milk  of 
the  mother  gave  like  results. 

LITERATURE. 

BRIEGER  and  ERLICH  :  "  Ueber  das  Auftreten 
des  Malignen  cedems  bei  Typhus  abdominalis," 


BACTEEIA    INVESTIGATION.  151 

Berliner  klin.  Wochenschr.  No.  43,  1882,  p. 
661. 

FLUGGE  :  "  Fermente  u.  Mikroparasiten "  in 
Zeimssen's  Handbuch  der  Hygiene,  Leipzig,  1883, 
p.  126. 

HEIBERG  :  (On  puerperal  fever) .  Leipzig, 
1873. 

KOCH  (R.):  "Bacillus  d.  Malignen  CEdems," 
Mittheil.  a.  des.  kais.  Gesundheitsamt,  1881. 

LAFFLER  (Tn.)  :  "  Gehirnerweichung  bedingt 
durch  Mikrokokken,  Infection  bei  puerperaler 
Pyaemia."  Breslauer  arztl.  Zeitschr.  1880,  p. 
205. 

MAYERHAFER  :  (Vibrio  of  puerperal  fever)  Mon- 
atsschrift  fur  Geburtskunde,  XXI. 

PASTEUR  (L.)  :  "Da  1'extension  de  la  theorie 
des  germs  a  1'etiologie  de  quelques  maladies  com- 
mune." Corapt.  Rend.  T.  90,  p.  1033  (1883)  II. 
"  Ueber  d.  Vibr.  septique,"  Bull,  de  1'acad. .  de 
med.,  1877. 

SEPTICAEMIA  OF  DOMESTIC  MOUSE. 

Bacillus  septicus  (Koch). 

This  form  of  septicaemia  is  also  known  as  Koch's 
septicaemia. 

Koch's  Method  of  cultivation  was  by  solid  media, 
either  a  mixture  of  aqueous  humor  and  gelatine, 
or  of  gelatine,  peptone  (1  per  cent),  salt  (0.6  per 
cent) ,  and  sodium  phosphate  in  sufficient  quantity 


152  THE  TECHNOLOGY   OF 

to  render  the  mass  alkaline  in  reaction.  The  Ba- 
cilli grow  well  on  this  mixture,  and  by  repeated 
and  rapid  division  form  peculiar  branched  series. 

SEPTICAEMIA  OF  RABBITS. 
Bacterium  septicaemias  (Koch). 

Koch  produced  this  disease  in  rabbits  by  inject- 
ing them  with  water  from  the  rivulet  Pauke,  and 
with  putrid  mutton  infusions.  The  microbe  seems 
to  be  the  same  as  that  of  "Davaine's  septicaemia," 
which  was  first  produced  by  injecting  rabbits  with 
putrid  ox  blood.  The  two  diseases  are  distin- 
guished in  that  Davaine's  septicaemia  is  easily 
transmissible  to  guinea-pigs,  but  not  to  birds ; 
while  mice,  pigeons,  fowls,  and  sparrows  are  very 
susceptible  to  the  disease  discovered  by  Koch, 
but  guinea-pigs,  dogs,  and  rats  resist.  Koch 
says  that  upon  examining  more  closely  the  "  new 
disease "  described  by  Pasteur,  as  being  induced 
by  a  child  dead  of  hydrophobia,  it  was  found 
to  be  "  the  already  long-known  septicaemia  of 
rabbits."  This  should,  however,  be  further  investi- 
gated. 

This  Bacterium  stains  in  such  a  manner  that  be- 
tween the  intensely  colored  poles  a  portion,  about 
one  half  of  the  entire  length,  remains  unstained. 
This  gives  the  microbes  an  apparent  dumb-bell 
shape,  and  they  are  easily  mistaken  for  two  mi- 
crococci. 


BACTERIA    INVESTIGATION.  153 


LITERATURE. 

DOWDESWELL  (G.  F.)  :  "On  the  action  of  heat 
upon  the  contagium  in  the  two  forms  of  septicae- 
mia known  respectively  as  '  Davaine's '  and  '  Pas- 
teur's.'" Proc.  Roy.  Soc.,  1882.  No.  221. 

GAFFKY  :  Mittheilung  aus  dem  Reichs-Gesund- 
heitsamt.,  Bd.  1,  1881,  p.  97. 

KOCH  (R.)  :  "Bacillus  d.  Septicaemie  bei  Mau- 
sen."  Ibid. 

SYPHILIS. 

Bacillus  KlebsiL 

Syphilis  furnishes  another  example  of  a  disease 
having  two  claimants  to  the  position  of  materias 
morbi,  a  Micrococcus,  and  a  Bacillus. 

Birsch-Hirschfeld's  Method:  Make  sections  of 
syphilitic  lesions  which  have  been  hardened  in  ab- 
solute alcohol.  Stain  these  in  a  concentrated 
aqueous  solution  of  fuchsin,  obtained  by  thinning 
an  alcoholic  solution  of  the  dye ;  wash  in  distilled 
water  ;  dehydrate  with  absolute  alcohol ;  clear  with 
oil  of  cloves,  and  mount  in  Canada  balsam. 

Klebs'  Method  of  inoculation  was  to  cut  out 
pieces  of  syphilitic  lesions,  using  all  precautions, 
carbolic  acid  solution  spray,  etc.  ;  pure  cultures 
were  then  made  from  these  pieces,  and  apes  were 
inoculated  with  the  cultures.  These  animals 
showed  typical  syphilitic  lesions  as  a  result  of 
such  inoculation. 


154  THE  TECHNOLOGY   OF 


LITERATURE. 

AUFRECHT  :  "  Ueber  den  Befund  von  Syphilis- 
Mikrokoken."  Ctbl.  f.  d.  med.  Wiss,  1881.  No. 
13,  p.  228. 

BmcH-HiRSCHFELD  (F.  N.)  :  "  Bakterien  in 
syphilitischen  Neubildungen."  Ctbl.  f.  d.  med. 
Wiss.  1882,  No.  33.  Abstr.  in  Deutsch.  med. 
Wochenschr.  1882,  p.  505. 

HEYDEN  (W.  H.  VAN  DER)  :  "Preservation  de 
la  syphilis  par  la  vaccin.  Traitement  des  maladies 
infectieuses,  Hypothese  sur  le  r61e  des  microbes 
dans  la  formation  des  animaux."  Transl.  from 
Dutch  by  A.  E.  Roberts,  8°  IV.,  51  pp.,  Utrecht 
(J.  L.  Beijers),  1883. 

KLEBS  (E.)  :  "  Das  contagium  der  Syphilis. 
Eine  experimentelle  Studie."  Archiv.  f.  Exp. 
Pathol.,  etc.,  X.,  p.  161.  II.  «  Bakterien  in  frisch 
extirpirten  syphilitischen  Primarindurationen." 
Archiv  f.  exper.  Pathol.  Bd.  X.  Hft.  3  u.  4,  p. 
161-221. 

LUSTGARTEN  (S.) :  "  Ueber  specifische  Bacillen 
in  syphilitischen  Krankheitsproducten."  (Vorl. 
Mitt.)  Wiener  med.  Wochenschr,  1884.  No.  49. 

MARTINEAU  ET  HARMONIE  :  "De  la  bacteridie 
syphilitique ;  de  1'evolution  syphilitique  chez  le 
pore."  Gaz.  hebdomad.  1882,  8  Sept.,  p.  589. 
Abstr.  in  Deutsch.  med.  Wochenschr.,  1882,  p. 
670. 


BACTERIA    INVESTIGATION.  155 

MORI  SON  :  I.  "  Ueber  das  Vorkommen  von 
Bakterien  bei  Syphilis."  Wiener  Med.  Wochen- 
schr.  1883.  No.  3.— II.  "Ueber  das  Vorkommen 
von  Bakterien  in  syphilitischen  Secreten."  Prager 
med.  Wochenschr.  1883.  No.  13. 

STRAUS  (I.)  ;  "  Sur  la  virulence  du  bubon  qui 
accompagne  le  chancre  mou."  Compt.  Rend.  T. 
99,  Nov.  24,  1884,  p.  935. 

TUBERCULOSIS. 
Bacillus  tuberculosis  (Koch). 

The  property  of  retaining  their  color  against  the 
action  of  strong  acids,  after  having  been  once 
stained,  is  characteristic  of  tubercle  Bacilli,  and  of 
none  other,  so  far  as  is  known,  with  the  single  ex- 
ception of  those  of  leprosy.  We  may  be  certain 
of  our  diagnosis  when  we  find  the  Bacilli  retaining 
their  color  after  being  exposed  to  the  action  of  acid 
solutions.  It  is  often  the  case  that  the  stained  tu- 
bercle Bacilli  show  uncolored  portions  of  a  round 
or  oval  form,  which  are  regarded  as  spores.  I 
have  given  below,  for  the  sake  of  completeness,  all 
the  so-called  "'new  methods,"  which  are  mostly 
only  modifications  of  Ehrlich's  method,  which  for 
diagnosis  answers  every  requirement,  and  is  ac- 
cepted as  the  best  by  Koch,  Friedlander,  etc.1  Dr. 

1  In  the  employment  of  any  of  the  methods  given  for  spu- 
tum, it  is  very  important  that  the  layer  of  sputum  should  not 
he  too  thick,  —  about  such  a  layer  as  one  uses  when  studying 
blood.  Make  about  six  sputum  preparations  at  once. 


156  THE  TECHNOLOGY  OF 

Koch  originally  held  that  tubercle  Bacilli  would 
only  take  staining  when  simultaneously  exposed  to 
the  action  of  an  alkali.  His  original  method  of 
procedure  was  as  follows  :  — 

Koch's  Method.  —  The  material  to  be  studied  is 
prepared  according  to  general  rules  for  "  cover- 
glass  preparations,"  or  sections,  and  placed  for 
from  twenty  to  twenty-four  hours  in  an  alkaline 
methyl-blue  solution  [F.  23.] .  The  time  may  be 
shortened,  if  desirable,  to  one  hour  by  heating  in 
a  drying-oven  at  40°  C.  Wash  the  stained  prepa- 
rations in  water,  and  place  them  in  a  filtered  con- 
centrated aqueous  solution  of  vesuvin  for  one  or 
two  minutes  for  sputum,  and  fifteen  to  twenty 
minutes  for  sections,  then  wash  again  in  distilled 
water  until  all  blue  color  disappears  and  a  more  or 
less  deep  brown  color  is  left.  Under  the  micro- 
scope all  portions  of  the  tissue,  especially  the  nu- 
clei, and  any  micrococci  or  products  of  decompo- 
sition which  may  be  present,  will  be  found  stained 
brown,  while  the  tubercle  Bacilli  are  a  beautiful 
blue.  Dr.  Koch  says  :  "  All  other  Bacilli,  except 
lepra  Bacilli,  which  I  have  investigated,  take  a 
brown  color  in  this  method."  After  staining,  de- 
hydrate with  absolute  alcohol,  clear  up  with  oil  of 
cloves,  and  mount  in  Canada  balsam.  The  above 
method  is,  however,  an  unsatisfactory  one,  since 
the  Bacilli  mz  hard  to  find,  and  the  sputum  prepara- 
tions are  not  good  on  account  of  the  solubility  of 


BACTERIA    INVESTIGATION.  157 

the  mucin  in  the  staining  fluid.  It  was  improved, 
and  a  good  substitute  found  for  the  caustic  potash 
in  the  so-called  "  aniline  oil "  [F.  4.]  in  — 

Wirlich's  Method  (now  recommended  by  Dr. 
Koch),  which  was  based  upon  the  hypothesis  that 
the  Bacilli  were  penetrable  to  staining  fluids  having 
a  low  alkaline  reaction,  but  that  they  were  sur- 
rounded by  an  envelope  impenetrable  to  acids. 
Erlich  sustained  this  opinion  by  subjecting  his 
preparations  to  a  solution  of  nitric  acid,  by  which 
the  tissues  were  entirely  bleached,  while  the  Ba- 
cilli retained  their  color. 

For  sputum  he  pressed  a  particle  between  two 
cover-glasses  by  means  of  a  preparation  needle, 
and,  sliding  them  apart,  passed  them,  prepared  sur- 
face up,  through  the  gas  flame.  He  then  allowed 
them  to  float,  prepared  side  down,  in  a  watch-glass 
filled  with  gentian-violet,  methyl- violet,  or  fuchsin- 
anilin-oil  solution  [F.  17.]  for  fifteen  minutes  to 
half  an  hour.  By  heating  the  whole  over  the  flame 
until  it  steams,  they  need  only  be  left  for  one  min- 
ute. Wash  in  a  solution  of  nitric  acid  (33^  per 
cent),  under  the  influence  of  which  the  color  soon 
fades  out  in  the  matrix,  the  Bacilli  alone  retaining 
the  color  —  violet  or  red.  Wash  in  distilled  water, 
dry,  pass  through  absolute  alcohol,  and  mount  in 
Canada  balsam.  A  better  and  clearer  picture  is 
obtained  by  making  a  double  staining,  i.  e.,  re- 
staining  the  decolorized  matrix.  This  is  best 


158  THE  TECHNOLOGY   OF 

accomplished  by  means  of  some  complementary 
color,  e.  g.,  aqueous  solutions  of  Bismarck-brown, 
vesuvin,  or  malachite-green.  Ehrlich's  method 
excelled  that  proposed  by  Koch  in  its  greater  ra- 
pidity and  in  the  large  number  of  Bacilli  colored. 
In  the  discussion  following  its  announcement  Dr. 
Koch  said  he  preferred  it  to  his  own,  and  now  uses 
it  altogether.  Its  disadvantages  are,  however,  that 
many  Bacilli  are  decolorized. 

For  sections.  —  Thin  sections  must  remain  in  the 
staining  fluid  for  twenty- four  hours,  and  for  two 
or  three  minutes  in  the  acid  mixture.  Wash  well 
in  repeatedly-renewed  water  (hereupon  depends 
the  tenacity  of  the  Bacilli  staining) ,  pass  through 
absolute  alcohol,  oil  of  bergamot  or  cloves,  and 
mount  in  Canada  balsam.  Some  months  after  Ehr- 
lich  published  the  above  modification  of  Koch's 
first  method,  Ziehl  opposed  the  statement  that  the 
external  envelope  of  tubercle  Bacilli  is  only  pene- 
trated by  coloring  matter  when  under  the  influence 
of  alkalies,  and  substituted,  for  aniline  oil  and 
caustic  potash,  carbolic  acid,  according  to  the  fol- 
lowing plan :  — 

ZiehTs  Method.  —  I.  Prepare  as  in  Ehrlich's 
method,  but  omit  the  decolorizing  with  the  nitric 
acid  solution,  which  drives  out  the  coloring  matter 
from  all  the  Schizomycetes  with  the  exception  of 
the  tubercle  Bacillus,  thus  allowing  the  working  of 
the  methyl-blue  solution.  II.  Prepare  exactly  as 


BACTERIA    INVESTIGATION.  159 

in  Ehrlich's  method,  but  in  place  of  the  aniline  oil 
use  resorcin,  pyrogallic  acid,  or  carbolic  acid.  In 
this  manner  he  demonstrated  that  the  tubercle  Ba- 
cilli did  not  need  an  alkaline  staining  fluid. 

Still  later  Dr.  Prior  showed  that  —  (1)  Ehrlich's 
staining  fluid  is  not  alkaline,  but  neutral ;  (2)  that 
the  staining  of  tubercle  Bacilli  is  successful  if  a 
neutral,  alkaline,  or  weakly  acid  fluid  be  used  ;  (3) 
that  it  is  successful  if  a  non-alkaline  fluid,  e.  g.9 
turpentine  oil,  takes  the  place  of  the  aniline  oil. 
The  next  modification  proposed  was  the  — 

Balmer  and  FranzeVz  Method.  —  Prepare  as  for 
other  methods,  and  allow  the  cover-glasses  to  float 
for  twenty-four  hours,  prepared  side  down,  upon 
a  quantity  of  aniline-gentian-violet  solution  [F. 
19ft].  Then  wash  in  acidulated  water  [F.  7.]  for 
one  half  to  one  minute,  or  until  the  color  disap- 
pears, wash  in  distilled  water,  and  color  the  ground 
with  a  concentrated  aqueous  solution  of  Bismarck- 
brown  [F.  2.].  This  method  has  the  advantage 
that  few  Bacilli  are  overlooked,  but  it  takes  too 
long,  and  is  inconvenient,  as  it  requires  the  stain- 
ing fluid  to  be  made  new  each  time. 

In  view  of  this  inconvenience,  and  to  afford  a 
method  which  physicians  could  use  to  advantage 
for  diagnosis,  Rindfleish  taught  his  pupils  the  fol- 
lowing method :  — 

Rindfleish's  Method.  —  Pour  into  a  test-tube 
sufficient  aniline  oil  to  fill  the  fundus,  then  add 


160  THE  TECHNOLOGY  OF 

water  until  it  is  a  third  full,  shake  it  well,  and  filter 
through  a  small  filter,  that  can  be  held  in  the  hand, 
into  another  test-tube.  To  the  clear  filtrate  add 
eight  drops  of  a  concentrated  alcoholic  solution  of 
fuchsin  [F.  16.].  Now  place  before  you  upon  a 
piece  of  white  paper —  (1)  a-  watch-glass  half  full 
of  alcohol,  to  which  add  two  drops  of  dilute  nitric 
acid;  (2)  another  watch-glass  half  full  of  the 
above  prepared  fuchsin  solution ;  (3)  a  lighted 
spirit  lamp.  Now  with  the  pincers  take  up  the 
cover-glass  holding  the  dried  sputum,  and  pass  it 
three  times,  with  the  sputum  side  up,  through  the 
flame  ("about  as  fast  as  you  would  cut  bread"),  thus 
rendering  the  albumen  homogeneous.  Now  lay  the 
cover-glass,  prepared  side  down,  upon  the  staining 
fluid,  and  with  the  pincers  pick  up  this  watch-glass 
and  hold  it  over  the  flame  until  it  begins  to  steam, 
then  remove  the  cover-glass,  and  after  washing  it 
in  a  stream  of  distilled  water,  place  it  in  the  acidu- 
lated alcohol,  allow  it  to  remain  here  until  the  vio- 
let clouds  fade  out.  In  from  ten  to  fifteen  seconds 
it  will  appear  to  be  stained  only  at  a  few  points ; 
now  wash  it  in  distilled  water,  dry  it,  pass  it 
through  absolute  alcohol,  oil  of  cloves,  and  mount 
in  Canada  balsam.  For  examination  use  your 
highest  powers  and  remove  diaphragms. 

Orth's  Method  was  to  use  water  acidulated  with 
hydrochloric  acid  [F.  7]  in  place  of  the  usual 
nitric  acid  water,  and  to  stain  with  picro-carmine 


BACTERIA    INVESTIGATION.  161 

in  place  of  vesuvin.  This  gives  the  Bacilli  blue, 
and  the  nuclei  red.  Stain  first  in  gentian- violet, 
wash  in  distilled  water,  transfer  to  picro-carmine, 
then  to  hydrochloric  acid  alcohol ;  wash  in  pure 
alcohol,  and  mount  in  damar.  Never  use  Canada- 
balsam  dissolved  in  chloroform. 

The  next  method  is  not  a  good  one  for  diag- 
nostic purposes,  because  the  peculiar  relation  of 
tubercle  Bacilli  to  coloring-matter  is  not  based 
upon  taking  on  of  color,  but  only  upon  the  power 
which  they  possess  of  retaining,  to  a  certain  degree, 
this  color,  when  it  is  once  taken  on,  against  the 
effect  of  mineral  acids.  If  one  carries  on  the 
decolorizing  process  under  the  microscope,  he  will 
note  that  upon  the  addition  of  nitric  acid  all  the 
color  disappears  from  the  Bacilli,  but  that  upon 
the  addition  of  water  it  returns. 

Lichtheim's  Method.  Prepare  according  to 
general  rules.  The  staining  fluid  used  is  a 
concentrated  aqueous  solution  of  fuchsin  or  gen- 
tian-violet, made  by  adding  some  of  the  alcoholic 
solution  to  some  distilled  water.  .Allow  the  cover- 
glasses  to  float  in  this  for  thirty-six  hours.  The 
time  may  be  shortened  by  warming.  The  Bacilli 
do  not  stain  so  strongly  as  by  other  methods ; 
wash  in  water,  absolute  alcohol,  oil  of  cloves,  and 
Canada  balsam.  The  next  modification  is  — 

Petri's  Method.  I.  Petri,  like  Ziehl,  considered 
the  use  of  aniline  oil  as  superfluous.  Prepare 


162  THE   TECHNOLOGY   OF 

clear  filtered  solutions  of  fuchsin  and  malachite- 
green  in  alcohol,  thin  5  ccm.  of  each,  with  100 
ccm.  of  distilled  water.  The  cover-glasses  are 
prepared  according  to  general  rules.  Cover  the 
fuchsin  solution  with  a  glass  plate,  and  heat  until 
the  cover  is  dimmed  with  .condensed  vapor,  then 
allow  the  prepared  cover-glasses  to  float,  prepared 
side  down,  upon  the  hot  fluid,  and  cover  it  again, 
allowing  it  to  stand  for  from  one  to  fifteen  minutes. 
Then  take  out  the  deeply  stained  preparations,  and 
place  them  in  a  glass  dish  having  a  lip ;  pour  over 
them  some  glacial  acetic  acid ;  in  a  few  minutes 
they  will  be  sufficiently  decolorized.  If  the  acetic 
acid  is  made  very  red,  wash  them  a  second  time  in 
some  fresh  acid.  Then  wash  them  three  or  four 
times  with  water,  by  decanting,  in  the  same  dish, 
and  finally  pour  the  malachite-green  solution  over 
them.  After  from  five  to  ten  minutes  they  will 
be  sufficiently  stained.  Now  wash  in  water,  dry 
upon  filter-paper  placed  on  a  warm  surface.  Mount 
in  glycerine  or  Canada  balsam,  and  examine  with 
a  hydrate  of  chloral  immersion  objective.  This 
is  better  than  an  oil  immersion,  because  the  prepa- 
ration is  easily  washed  by  pouring  water  over  it. 
The  Bacilli  will  be  stained  red,  the  other  parts  of 
the  sputum  blue-green,  and  they  keep  color  better 
than  when  mineral  acids  are  used. 

II.  Instead  of  fuchsin  and  malachite-green,  ani- 
line-gentian-violet [F.  19]  and  aniline  yellow  [F.6] 


BACTERIA    INVESTIGATION.  163 

may  be  used.  Allow  the  sections  to  remain  in  a 
fresh  solution  of  the  aniline-gentian-violet  for 
half  an  hour,  then  for  eighteen  hours  in  20  ccrn. 
of  absolute  alcohol,  rinsing  two  or  three  times ; 
wash  for  one  minute  in  distilled  water,  and  place 
in  an  aqueous  solution  of  aniline  yellow  for  three 
minutes.  Wash  in  absolute  alcohol,  pass  through 
oil  of  cloves,  and  mount  in  Canada  balsam.  The 
advantages  of  this  method  are  the  large  number 
of  Bacilli  stained,  and  the  permanence  of  the 
staining.  Dr.  Plaut,  of  Leipzig,  had  preparations 
made  by  this  method  remain  good  for  over  a  year, 
and  recommends  it  for  dealers  in  microscopical 
preparations. 

Gibbs1  Method.  Prepare  according  to  general 
rules.  Allow  the  preparations  to  remain  for  from 
fifteen  to  twenty  minutes  in  a  solution  of  magenta 
[F.  26].  Then  wash  in  an  aqueous  nitric  acid 
mixture  (1  part  to  3),  and  afterwards  in  alcohol. 
Give  a  double  color  with  chrysoidine  [F.  11], 
Wash  in  alcohol,  pass  through  oil  of  cloves,  and 
mount  in  Canada  balsam. 

GibM  New  Method.  Allow  the  prepared  cover- 
glasses  to  float  for  five  minutes  in  a  solution  of 
Eosalin-chlorhydrate  methyl-blue  [F.  36.  23], 
which  has  been  warmed  to  steaming.  Wash  in 
methylic  alcohol  until  no  more  coloring  matter  is 
given  off.  Dry ;  pass  through  absolute  alcohol, 
oil  of  cloves,  and  mount  in  Canada  balsam. 


164  THE  TECHNOLOGY  OF 

Sections  must  remain  several  hours  in  the  stain- 
ing fluid,  otherwise  they  are  handled  the  same  as 
cover-glass  preparations.  The  advantages  ot  this 
method  are,  that  on  account  of  no  acids  having 
been  used  there  is  no  shrinking  of  the  tissue,  and 
the  preparations  retain  their  color  very  well.  Ac- 
cidental Bacteria,  in  the  neighborhood  of  the 
Bacilli ',  will  often  be  found  stained  red.1  The 
above  is  a  method  which  is  strongly  recommended, 
as  is 

Baumgarteris  Method  (for  sputum).  Prepare 
according  to  general  directions  for  cover-glass 
preparations,  and  moisten  the  dried  preparations 
in  a  watch-glass  of  distilled  water,  to  which  has 
been  added  one  or  two  drops  of  a  solution  of 
caustic  potash.  By  the  use  of  a  power  of  four 
or  five  hundred  diameters  the  Bacilli  are  already 
recognizable.  They  do  not  appear  any  larger 
than  those  prepared  by  the  aniline  coloring  meth- 
ods. To  avoid  confusing  them  with  other  Schizo- 
mycetes,  drop  upon  the  dried  cover-glass  a  drop 
of  aqueous  aniline-violet  solution.  The  tubercle 
Bacilli  appear  absolutely  colorless.  This  process 
does  not  give  good  results  for  diagnosis,  but  is  a 
convenient  method  of  rendering  the  Bacilli  visible 

o 

1  Drs.  Baumgarten  and  Frankel  claim  that  frequently  the 
accidental  Bacteria  take  the  blue  color,  while  the  Bacilli  are 
stained  red,  thus  rendering  the  method  useless  for  diagnosis.  — 
Yid.  Zeitschr.  f.  wiss.  Mikroskopie,  Bd.  1,  Heft.  3,  p.  457. 


BACTERIA    INVESTIGATION.  165 

without  coloring,  and  is  recommended  especially 
for  demonstration,  as  it  shows  that  the  tubercle 
Bacilli  behave  differently  from  most  other  forms 
of  Bacilli  in  being  decolorized,  as  well  as  the 
nuclei  of  the  tissue.  This  also  happens  if  they 
are  prepared  according  to  Gram's  general  method. 

Baumgarterts  New  Method.  In  a  small  watch- 
glass  full  of  distilled  water  drop  four  or  five  drops 
of  concentrated  alcoholic  solution  of  methyl-violet. 
Allow  the  preparations  to  become  deeply  stained 
in  this  fluid ;  wash  in  water  for  from  five  to  ten 
minutes,  and  pass  through  absolute  alcohol ;  then 
lay  them  in  a  half-saturated  solution  of  carbonate 
of  potassium,  whereby  the  tissue  is  completely 
decolorized.  Wash  in  absolute  alcohol,  pass 
through  oil  of  cloves,  and  mount  in  a  mixture  of 
equal  parts  of  oil  of  cloves  and  Canada  balsam 
(free  from  chloroform). 

(For  sections.)  Stain  as  for  cover-glass  prepa- 
rations, then  place  for  five  minutes  in  alcohol, 
and  afterwards  in  an  acetic  acid  solution  of  Bis- 
marck-brown ;  finish  as  for  cover-glass  preparations, 
accidental  bacteria  appear,  according  to  this  pro- 
cess, brown ;  if  numerous,  covering  up  the  often 
sparsely  present  tubercle  Bacilli.  The  use  of  the 
carbonate  of  potash  solution  is  also  to  be  recom- 
mended here,  before  using  the  Bismarck-brown. 
In  place  of  the  latter,  borax  carmine  or  borax- 
picro-carmine  may  be  used. 


166  THE   TECHNOLOGY   OF 

Baumgarten's  Culture  Method. — Take  from  a 
living  animal  a  small  fragment  of  a  Tubercle 
nodule  (or  from  a  perfectly  fresh  human  nodule, 
rich  in  Bacilli),  and  introduce  it  with  all  the 
proper  antiseptic  precautions  into  the  anterior 
chamber  of  the  eye  of  a  living  rabbit.  Here  the 
piece  increases  in  size,  owing  to  the  development 
of  the  Bacilli.  Allow  it  to  remain  for  six  or  eight 
days,  and  then  remove  it  and  take  a  small  particle 
from  it.  Place  this  second  particle  in  the  anterior 
chamber  of  the  eye  of  another  living  rabbit,  allow 
this  to  remain  six  or  eight  days,  and  place  a  third 
particle  in  the  eye  of  a  third  rabbit.  Proceed  in  this 
way  until  an  absolutely  pure  culture  is  obtained. 
This  method  has  the  advantage  over  the  methods 
of  pure  culture  outside  the  living  body,  with  arti- 
ficial culture  apparatus,  that  it  requires,  with  the 
exception  of  the  antiseptic  precautions  of  the  oper- 
ation, no  further  trouble  with  sterilization  or  regu- 
lation of  temperature. 

Weigerfs  Method.  Prepare  according  to  gen- 
eral rules,  and  stain  in  gentian-violet  solution 
[F.  18.] ,  and  handle  further  according  to  Ehrlich's 
method.  The  advantages  of  this  method  are,  that 
it  renders  visible  the  Bacteria  of  a  section  when 
present  in  very  slight  numbers,  and  is  to  be 
recommended  for  use  in  tuberculosis  of  men  and 
cattle. 

FrdnkeTs  Method,  for  the  rapid  double  staining 


BACTERIA    INVESTIGATION.  167 

of  sputum.  To  5  ccm.  of  aniline- water  [F.  5.], 
heated  to  100°  C.,  add,  drop  by  drop,  a  saturated 
solution  of  methyl- violet  or  fuchsin,  until  a  strong 
opalescence  is  produced.  Allow  the  cover-glasses 
to  float  upon  the  heated  fluid  for  two  minutes,  and 
then  decolorize,  and  re-stain  simultaneously  in  an 
(herein  lies  the  peculiarity  of  the  method)  acid 
alcoholic  solution  of  a  contrasting,  complementary 
dye.  (1.)  For  blue,  use  alcohol  50  parts,  water 
30,  hydrochloric  acid  20,  and  as  much  methyl 
blue  as  will  dissolve  after  repeated  shaking ;  filter. 
(2.)  For  brown,  alcohol  70  parts,  hydrochloric 
acid  30,  as  much  Vesuvin  as  it  will  dissolve  ;  filter. 
(3.)  For  green,  alcohol  50  parts,  water  20,  acetic 
acid  30,  and  as  much  malachite  or  methyl-green 
as  It  will  dissolve ;  filter.  Lay  the  stained  cover- 
glass  in  one  of  these  prepared  solutions  (which 
may  be  kept  on  hand)  for  one  or  two  minutes, 
wash  in  water  or  dilute  acid  (1  per  cent  acetic), 
then  in  50  per  cent  alcohol,  dry,  first  between 
blotting-paper,  then  over  the  flame.  In  four  min- 
utes a  good  double- stained  preparation  is  made, 
which  is  permanent. 

Pfuhl  Petri's  Method.  Prepare  according  to 
general  directions,  and  stain  in  a  solution  of  fuch- 
sin [F.  16.]  by  floating  the  cover  glasses  in  it  for 
one  or  two  minutes,  warm  to  steaming  in  the  mean- 
time. Decolorize  in  acetic  acid,  wash  in  water. 
Give  a  second  staining  by  means  of  a  solution  of 


168  THE  TECHNOLOGY   OF 

malachite  green  [F.  24]  thirty  seconds  to  one 
minute,  wash  in  water,  pass  through  absolute 
alcohol,  oil  of  cloves,  and  mount  in  Canada 
balsam. 

Senkewitsch's  Method.  Prepare  according  to 
general  directions,  and  place  in  a  concentrated  so- 
lution of  fuchsin.  After  the  preparations  are 
sufficiently  stained,  wash  for  one  or  two  minutes  in 
10  cc.  of  alcohol,  to  which  one  drop  of  nitric  acid 
has  been  added.  Wash  in  water,  dry,  pass 
through  absolute  alcohol,  oil  of  cloves,  and  mount 
in  Canada  balsam. 

Kddtzers  (combined)  Method.  Spread  out  a 
quantity  of  sputum  upon  a  black  plate,  and  pick 
out  the  opaque  white  or  gray-white  patches  by 
means  of  two  previously  sterilized  needles.  Pre- 
pare cover  glasses  according  to  general  directions, 
and  stain  in  a  filtered  solution  of  aniline-gentian- 
violet  [F.  18]  by  allowing  the  cover-glasses  to 
float  for  twenty-four  hours  or  more  in  the  same, 
or  hasten  matters  by  heating  up  to  80°  C.  for  sev- 
eral minutes,  remove  and  soak  up  the  excess  of 
staining  fluid  on  the  cover  glasses  by  means  of  a 
bit  of  blotting  paper,  then  place  in  a  watch  glass 
of  acidulated  alcohol  [F.  7]  for  from  one  half  to 
one  minute,  then  remove  to  a  watch-glass  contain- 
ing 90  per  cent  alcohol,  until  the  color  has  faded 
out,  then  wash  in  distilled  water.  Now  drop  upon 
the  preparation  four  or  five  drops  of  a  concen- 


BACTERIA    INVESTIGATION.  169 

trated  aqueous  solution  of  vesuvin.  After  two 
minutes,  wash  in  distilled  water,  dry,  pass  through 
absolute  alcohol,  oil  of  cloves,  and  mount  in  Can- 
ada balsam  or  damar  varnish. 

Long's  Method  for  Sputum.  The  entire  quan- 
tity of  sputum  to  be  investigated  is  placed  in  alka- 
line water.  For  this  purpose  a  watch-glass  is 
used,  into  which  is  poured  five  or  six  grammes  of 
distilled  water,  and  to  this  are  added  three  or  four 
drops  of  a  33  per  cent  caustic-potash  solution  by 
means  of  a  burette ;  after  this  is  well  mixed  the 
sputum  is  placed  in  it.  In  about  half  an  hour  the 
latter  is  much  dissolved,  the  air-bubbles  much 
diminished,  and  grayish-green  stripes  may  be  seen 
in  the  compact  masses,  which,  if  Bacilli  are  pres- 
ent, are  sure  to  contain  them.  By  this  method 
small  clear-white  scales  are  seen  which  resemble 
the  culture  colonies  as  described  by  Dr.  Koch, 
which  they  really  are.  Stain  according  to  Balmer- 
FranzeFs  Method,  and  use  for  demonstration  an 
Abbe's  condenser,  and  a  one-twelfth  homogeneous 
immersion  objective. 

Peter's  Method  for  Sections,  to  stain  all  the 
bacilli  present.  Thin  sections,  which  have  been 
hardened  in  alcohol,  are  placed  for  one  moment  in 
distilled  water,  then  for  thirty  minutes  in  a  two  per 
cent  aqueous  gentian-violet  solution  (filtered  and 
made  with  fresh  aniline-water).  Then  place  for 
eighteen  hours  in  at  least  twenty  grammes  of  abso- 


170  THE  TECHNOLOGY   OF 

lute  alcohol  in  a  closed  vessel,  renewing  the  alco- 
hol once  or  twice.  Wash  for  one  minute  in  dis- 
tilled water,  then  place  for  three  minutes  in  a  two 
per  cent  aqueous  solution  (filtered)  of  aniline- 
yellow,  then  from  five  to  thirty  minutes  in  absolute 
alcohol.  By  the  shorter  use  of  absolute  alcohol  the 
Bacilli  are  easier  found,  and  the  method  of  group- 
ing more  apparent ;  by  the  longer  use  the  struc- 
ture of  the  tissue  surrounding  the  Bacilli  is  made 
more  evident.  Pass  through  oil  of  cloves  and 
mount  in  Canada  balsam. 

Veraguth's  Method.  This  method  is  a  modifi- 
cation of  Ehrlich's,  proposed  in  order  that  the 
large  number  of  pathological  preparations  in  col- 
lections might  not  be  lost.  By  its  use  tubercle 
Bacilli  can  be  shown  in  old  chromic  acid  prepara- 
tions as  readily  as  in  freshly  hardened  alcoholic 
preparations.  Lay  small  pieces  of  the  preparation 
to  be  examined  for  two  or  three  days  in  flowing 
water,  and  then  place  in  alcohol.  When  ready  to 
cut,  place  them  for  twenty-four  hours  in  water, 
and  then  cut  with  a  freezing  microtome.  For 
pieces  of  lung,  use  with  this  instrument  a  solution 
of  gum  arabic  in  glycerine.  Before  staining,  place 
the  sections  for  twenty-four  hours  in  absolute  alco- 
hol, and  then  stain  in  aniline- water- fuchsin  for 
forty-eight  hours.  Decolorize  with  nitric  acid 
water,  and  wash  well  in  distilled  water.  Give 
double-staining  with  an  aqueous  solution  of  methyl- 


BACTERIA  INVESTIGATION. 

blue  for  from  five  to  ten  minutes,  wash  in  water, 
pass  through  absolute  alcohol,  oil  of  cloves,  and 
mount  in  Canada  balsam. 

Coze  and  Simon's  Method  for  Sputum. — The 
process  is  the  same  for  the  examination  of  all 
liquids,  only  when  the  liquids  are  insufficiently 
albuminous  to  adhere  to  the  glass,  a  few  drops  of 
fresh  white  of  egg  is  added,  which  gives,  by  its 
coagulation,  more  fixity  to  the  preparation.  Pre- 
pare cover-glasses  according  to  general  directions, 
and  place  in  a  solution  of  fuchsin  or  gentian-violet. 
(F.  19,  b.)  Heat  to  40°  C.  for  half  an  hour,  wash 
in  water,  pass  through  acid  solution  (F.  7.),  wash 
again  in  plenty  of  water,  and  place  for  two  or  three 
minutes  in  a  solution  of  chrysoidin  (F.),  wash  dry 
and  mount. 

For  Sections.  —  Harden  the  pieces  of  tissue  in 
absolute  alcohol  for  forty-eight  hours  or  more,  cut 
extremely  thin  sections,  and  place  them  for  twenty- 
four  hours  in  a  solution  of  fuchsin  (F.),  or  for 
one  hour  if  temperature  is  raised  to  40°  C.  Wash 
4-5  minutes  in  acid  solution  (F.),  then  in  water 
until  the  last  trace  of  acid  is  removed.  Then  place 
for  15-20  minutes  in  the  aniline  water  (F.  7. )  and 
afterwards  stain  in  chrysoidin  or  haematoxylin 
(F.  11.22.).  Wash,  dehydrate  in  absolute  alco- 
hol, pass  through  turpentine,  and  mount. 

Dejerine's  Method  for  studying  the  stony  con- 
crements  which  are  found,  about  the  size  of  a  pea,  in 


172  THE   TECHNOLOGY  OF 

the  apices  of  the  lungs  of  old  people,  was  to  pound 
them  up  in  a  mortar  with  a  little  distilled  water. 
In  the  limy  broth  he  found  fragments  of  organic 
substance  which  he  stained  after  Ehrlich's  method, 
and  in  which  he  found  tubercle  Bacilli.  These 
concretions  have  a  hard  nucleus  and  a  periphe- 
ral zone  of  the  consistence  of  poorly  hardened 
gypsum. 

Giboux's  Method,  to  test  the  inoculability  of  tu- 
berculosis through  respiration,  was  to  use  two 
boxes,  in  which  were  placed  rabbits,  and  through 
which  he  passed  daily  20,000-25,000  cm.  of  air 
that  had  been  exhaled  by  phthisical  patients.  One 
box  was  protected  by  having  the  air  filtered 
through  cotton.  After  three  and  a  half  months 
the  rabbits  in  the  unprotected  box  died,  and 
showed  in  their  lungs,  livers,  and  spleens,  tu- 
bercle Bacilli.  The  other  rabbits  remained 
healthy. 

Ermengerfs  Method.  —  Add  the  chosen  aniline 
dye  to  a  mixture,  of  aniline-oil  4  grammes,  alcohol 
20  grammes,  at  40°  C.,  to  this  add  an  equal  quan- 
tity of  distilled  water.  Prepare  fresh,  and  filter 
before  using.  Sulphate  of  rosanilin  and  methyl- 
violet,  quality  BBBBB,  preferred.  Decolorize 
the  preparations  in  dilute  nitric  acid,  and  wash 
thoroughly  in  distilled  water.  For  a  second  stain, 
use  aniline-blue,  aqueous  solution  of  vesuvin,  or, 
still  better,  'Grenacher's  carmine'  when  the  stain- 


BACTERIA    INVESTIGATION.  173 

ing  of  the  Bacilli  has  been  effected  by  means  of 
methyl-blue  or  -green.  Avoid  exposing  the  prepa- 
rations to  a  bright  light.  Mount  in  Canada  balsam, 
or  damar  dissolved  in  benzine,  which  preserves 
well  the  colors  produced  by  the  aniline  salts. 
Glycerine-jelly  may  also  be  used  successfully. 

Brun's  Method.  —  Spread  out  by  pressure  be- 
tween two  cover-glasses  a  particle  of  sputum,  and 
allow  it  to  remain  exposed  to  the  air,  but  pro- 
tected from  dust,  for  a  few  minutes.  The  thin, 
mucilaginous  layer  thus  obtained  contains  albu- 
men, mucin,  pus  granules,  fat  and  blood  cells, 
particles  of  carbon,  and  other  debris  of  respired  air. 
Drop  on  the  prepared  cover-glass  a  few  drops  of  a 
concentrated  solution  of  fuchsin  or  methyl-blue, 
(made  with  equal  parts  of  water  and  alcohol  con- 
taining l-30th  aniline  oil  to  render  it  alkaline) . 
Drop  the  cover-glass  into  a  test  tube  and  wash  in 
water  by  decanting,  then  wash  in  an  acidulated 
solution,  (F.  7.),  until  the  organic  matrix  is  very 
feebly  colored ;  wash  repeatedly  by  decanting  in 
the  same  test  tube  ;  then  cover  the  preparation  for 
a  few  minutes  with  a  concentrated  solution  of  ani- 
line oil ;  this  neutralizes  all  remaining  acid.  Mount 
in  fluid  (F.  43).  Keep  the  preparations  in  the 
dark.  The  advantages  claimed  for  this  method  are 
that :— (1)  It  avoids  heating  up  to  100°  or  120°  C., 
wrhich  coagulates  the  albumen  and  renders  it  opaque, 
besides  diminishing  the  size  of  the  microbes  by 


174  THE  TECHNOLOGY   OF 

contraction.  (2)  The  organic  matter  is  rendered 
transparent  by  means  of  acetic  acid.  (3)  The 
action  of  the  nitric  acid  remaining  in  the  organic 
matrix  is  neutralized  and  prevented  from  decolor- 
izing the  Bacteria  and  rendering  them  invisible 
after  a  time,  as  occurs  very  often  in  other  methods. 
(4)  Instead  of  Canada  balsam,  which  has  a  very 
high  refractive  index  (1.53),  a  neutral  liquid  hav- 
ing the  same  refractive  index  (1.37)  as  the  albu- 
minoid substances,  is  used. 

BurriWs  Method.  —  Not  finding  any  of  the  alco- 
holic solutions  of  aniline  dyes  satisfactory,  on  ac- 
count of  the  formation  of  a  precipitate  on  the 
cover-glasses  during  staining,  Prof.  B.  substitutes 
a  solution  of  fuchsin  in  glycerine  (F.  16).  Place 
the  cover-glass  in  this  solution  for  from  ten  min- 
utes to  several  days,  at  ordinary  temperatures. 
The  best  results  are  obtained  by  heating  up  to  80° 
or  100°  F.  for  25-30  minutes.  If  it  is  desired  to 
make  haste,  which  gives  less  good  results,  boil  a 
little  water  in  a  test  tube,  add  gradually  to  this 
double  its  quantity  of  the  above  staining  fluid, 
which  is  allowed  to  flow  down  the  side  of  the  tube 
held  obliquely,  shake  carefully,  turn  the  fluid  into 
a  watch-glass,  and  plunge  into  it  the  prepared 
cover-glass,  allow  it  to  remain  for  1—2  minutes, 
decolorize  in  a  solution  (1-4)  of  nitric  or  hydro- 
chloric acid,  wash  in  water,  dry,  and  mount. 

HartzeiVs  Method.  —  Prepare  cover-glasses  ac- 


BACTERIA    INVESTIGATION.  175 

cording  to  general  directions.  Place  upon  the 
dried  sputum 'one  or  two  drops  of  the  fuchsin  so- 
lution recommended  by  Gradle  (F.  16.),  and 
allow  it  to  remain  3-5  minutes,  decolorize  com- 
pletely in  oxalic  acid  solution,  wash  thoroughly  in 
water,  dry,  and  mount  in  glycerine  or  Canada 
balsam.  With  a  power  of  500  or*  600  the  Bacilli 
will  appear  as  brilliant  red  rods ;  no  staining  of  the 
background  is  necessary.  One  chief  advantage 
claimed  over  other  methods  is,  that  in  the  latter 
the  decolorizing  agent  employed  is  dilute  nitric 
acid,  but  this,  besides  being  disagreeable  to  handle 
because  of  its  staining  and  corrosive  qualities,  is 
apt  to  remove  the  color  from  the  Bacilli,  unless 
great  care  is  exercised.  Oxalic  acid,  however, 
seems  to  leave  the  dye  untouched. 

Quinlan's  Method.  —  Take  a  particle  of  the  ex- 
pectoration of  an  advanced  case  (the  first  expec- 
toration of  the  morning  is  preferable),  mix  it  with 
a  few  drops  of  a  solution  of  potash  by  means  of  a 
glass  rod,  until  it  is  homogeneous  ;  transfer  a  drop 
of  this  mixture  to  a  cover-glass,  and  dry  by  pass- 
ing rapidly  through  the  flame  of  an  alcohol  lamp. 
Place  upon  the  dried  sputum  a  few  drops  of  ma- 
genta (F.  26.),  allow  this  to  remain  for  twenty 
minutes ;  then  place  it  in  a  mixture  of  nitric  acid 
1  part,  distilled  water  2  parts,  until  it  is  bleached, 
which  will  be  in  about  five  minutes  ;  then  wash  in 
distilled  water.  Drop  upon  the  preparation  a  little 


176  THE  TECHNOLOGY   OF 

solution  of  methyl-blue,  allow  it  to  remain  five 
minutes,  wash,  and  dehydrate  in  absolute  alcohol. 
Place  a  drop  of  Canada  balsam  and  benzine  upon 
a  slide,  and  lay  the  prepared  cover-glass  upon  it. 
Bacilli  stained  scarlet  upon  a  blue  ground. 

Negri's  Method ',  for  staining  the  spores  in  the 
Bacilli  of  tuberculosis. 

(1)  Powdered  carmine gr.  0.5 

Strong  aqua  ammonia cc.  1 

Distilled  water "30 

Place  the  carmine  in  a  porcelain  capsule,  mix  it 
up  with  a  little  water,  add  the  ammonia  and  the 
remainder  of  the  water.  Allow  the  liquid  to  re- 
main in  the  air  protected  from  dust,  until  every 
trace  of  ammoniacal  odor  disappears,  this  is  essen- 
tial. Keep  it  at  a  temperature  of  about  15°  C.  for 
four  or  five  days.  Decant  the  clear  liquid  and 
throw  away  the  precipitate. 

(2)  Alcohol  (ordinary) cc.  100 

Hydrochloric  acid  (pure)     ....     drops  20 

(3)  Concentrated  solution  of  picric  acid  in  distilled  water, 
allow  an  excess  of  crystals  to  remain  at  the  bottom. 

(4)  Liquid  (2) 

(3)  of  each  15  cc. 

To  this  mixture  add  drop  by  drop,  stirring  all 
the  time,  the  solution  of  carmine  (1).  (Do  not 
add  the  acidulated  alcohol  to  the  carmine,  as  it 
gives  a  precipitate.)  Ordinarily  no  precipitate  is 
formed ;  if  it  does  form,  decant.  Add  a  small 


BACTERIA    INVESTIGATION.  177 

crystal  of  thymol,  and    preserve  in  a  stoppered 
bottle.     This  solution  Negri  calls  (5). 

(6)  Methyl-violet O.  gr.  7 

Absolute  alcohol cc.  10 

Aniline  oil 

After  complete  solution  of  the  coloring  matter 
add  distilled  water,  15  cc. 

The  methyl-violet  obtained  by  this  method  gives, 
according  to  Negri,  better  results  than  gentian-vio- 
let. It  is  necessary  to  have  a  good  methyl-violet, 
the  greater  part  of  the  violets  ordinarily  sold  not 
answering  the  purposes  of  the  microscopist. 

Spread  out  a  very  thin  layer  of  sputum  upon  a 
cover-glass  ;  do  not  heat  at  all,  or  only  very  slightly, 
as  too  much  heat  renders  the  Bacilli  difficult  to 
stain,  over-heating  is  probably  the  cause  of  much 
failure.  Place  the  cover-glass,  prepared-side  up, 
in  a  watch-glass,  and  drop  upon  it  the  solution  of 
the  aniline  dye  (6),  and  allow  it  to  remain  covered 
for  \-\  hour  or  more  at  a  temperature  of  15°  C. 
This  method  is  better  than  to  float  the  cover-glass 
upon  the  staining-fluid,  because  the  aniline  oil  is 
never  perfectly  dissolved,  and  forms  upon  the  top 
a  layer  which  prevents  the  staining  being  perfect 
and  intense.  Wash  the  cover-glass  in  plenty  of 
ordinary  water  until  all  excess  of  color  has  disap- 
peared;  then  plunge  it  into  the  acid  alcohol  (2), 
until  cleared,  wash  again  and  while  still  wet,  pour 
on  it  a  few  drops  of  the  carmine  solution  (5),  and 


178  THE  TECHNOLOGY  OF 

allow  it  to  stand  five  minutes.  Wash  off  the 
excess  of  carmine,  and  then  wash  again  in  the 
acid  alcohol  (2),  until  most  of  the  color  has  disap- 
peared ;  then  plunge  into  distilled  water,  which  is  to 
be  renewed  twice  in  the  course  of  8-10  minutes. 
Dry,  and  mount  in  pure  balsam,  by  heating  a  drop 
upon  a  slide. 

Upon  examination  one  sees  numerous  spores 
colored  azure-blue,  enclosed  in  the  transparent  en- 
velopes—  which  are,  however,  visible  if  observed 
with  care  —  of  the  Bacilli,  upon  a  rose-ground.  If 
it  is  desired  to  stain  the  Bacilli  entire  instead  of 
only  the  spores,  wash  the  preparation  after  the 
action  of  the  carmine  in  distilled  water,  without 
passing  through  the  acid  alcohol. 

Reinstadlefs  Culture  Method  for  tubercle  Bacilli. 
Place  a  piece  of  tubercular  lung  in  a  sterilized 
mortar  with  some  glass  sand  which  has  been  pre- 
viously heated  red  hot,  and  rub  the  mass  thor- 
oughly, or  comminute  the  lung  by  cutting  with 
scissors.  To  the  material  thus  obtained  add  a 
small  quantity  of  Bergmann's  fluid  [F.  1.]  which 
has  shortly  before  been  thoroughly  cooked  and 
cooled.  Mix  thoroughly  the  broth  thus  formed, 
and  filter ;  seal  up  for  further  use.  Several  test 
tubes  are  then  cleaned  by  boiling  them,  first  in 
nitric  and  sulphuric  acids,  and  then  in  alcohol. 
After  pouring  out  the  latter,  heat  them  in  the 
spirit  flame,  and  stop  the  perfectly  dry  tubes  with 


BACTERIA    INVESTIGATION.  179 

a  wad  of  carbolized  cotton.  Now  fill  them  by 
means  of  a  sterilized  pipette  with  about  30  cc. 
of  the  Bergmann's  fluid,  sterilize  the  whole  by 
heat,  and,  when  cool,  transplant  according  to  gen- 
eral rules  for  cultures. 

Celli  and  Guarnieri's  Method  for  ascertaining 
whether  the  microbes  of  tuberculosis  were  thrown 
into  the  air  by  the  breath  of  consumptives.  I.  For 
the  air  used  by  isolated  patients.  They  (C.  &  G.) 
lit  a  gas  jet  in  the  lower  part  of  a  large  tin  cylin- 
der, open  at  the  top  and  narrowing  down  below 
into  a  cone,  into  whose  apex  a  small  bent  tuba 
was  soldered,  whose  free  end  terminated  in  a  wide 
cone  of  tin,  into  which  was  introduced  a  cone  of 
copper  open  at  its  apex,  and  the  surface  of  which, 
after  having  been  heated  red  hot,  was  coated  with 
Koch's  gelatine.  The  warmth  of  the  gas  flame 
caused  a  current  of  air  to  pass  over  the  gelatine 
surface.  By  means  of  this  apparatus,  experiments 
were  made  for  twelve  nights  at  various  heights  in 
the  room,  the  usual  ventilation  of  which  was 
closed.  After  each,  the  gelatine  was  kept  at  an 
even  temperature  of  30°-40  C.  and  examined 
microscopically  by  Ehrlich's  method  ;  also  inocula- 
tions made.  II.  For  direct  expiration  of  con- 
sumptives. A  large  number  of  patients  were 
required  to  breathe  repeatedly,  throughout  twenty- 
four  hours,  (1)  partly  upon  a  small  dish  of  wood, 
with  a  concave  bottom,  coated  with  Koch's  gela- 


180  THE  TECHNOLOGY  OF 

tine,  and  covered  during  the  pauses  of  the  investi- 
gation with  a  watch-glass  ;  (2)  partly  into  a  glass 
tube  coated  inside  with  gelatine,  and  which  was 
only  open  during  the  investigation  ;  (3)  partly  in 
flasks  of  distilled  water  which  had  been  thoroughly 
sterilized,  and  through  the  corks  of  which  passed 
two  small  bent  glass  tubes,  the  short  one  being 
stopped  at  its  free  end  with  cotton,  the  patient 
breathed  into  the  longer  tube,  one  end  of  which 
dipped  into  the  fluid  ;  the  other  having  a  piece  of 
rubber  tubing  attached,  which  could  be  closed  with 
a  clamp ;  (4)  partly  in  a  Liebig's  ventilator  em- 
bedded in  ice,  the  entrance  and  exit  tubes  being 
arranged  as  in  (3).  The  gelatine  from  these  ex- 
periments was  investigated  by  Ehrlich's  method 
and  by  inoculations.  The  water  condensed  from 
the  breath  of  the  patients  by  apparatus  (4)  culti- 
vated in  sterilized  media.  III.  Sputum  was 
placed  in  a  retort  on  a  water  bath  at  34-40°  C.,  and 
the  vapor  thus  generated  was  collected  in  a  bulb 
packed  in  ice,  the  water  thus  obtained  was  exam- 
ined by  Ehrlich's  staining  method  and  also  placed 
in  culture  media.  Air  was  also  collected  from 
sputum  by  means  of  an  aspirating  apparatus.  The 
result  in  every  case  was  to  show  that  tuberculosis 
sputum,  so  long  as  it  is  moist,  does  not  give  out 
its  specific  Bacilli. 


BACTEETA    INVESTIGATION.  181 


LITERATURE. 

ARLOTNG  (S.)  :  Nouvelles  experiences  compara- 
tive sur  1'inoculabilite  de  la  scrofure  et  de  la  tuber- 
culose  de  1'horame  au  lapin  et  au  cobaye.  Compt. 
Rend,  de  1'Acad.  de  sc.  de  Paris,  T.  99,  Oct.  20, 
1884,  p.  661. 

AUFRECHT  :  "  Die  Aetiologie  der  Tuberculose," 
Centralblt.  f.  med.  Wissensch.  17,  1882,  also 
Deutsche  med.  Wochenschr.,  1882,  p.  283. 

BABES  :  "  Comparaison  entro  les  bacilles  de  la 
tuberculose  et  ceux  de  la  lepra  (Elephantiasis  des 
Grecs)."  Compt.  Rend.  T.  96,  p.  1244  and  1323 
(1883). 

BALMER  UND  FRAENTZEL  :  "  TJeber  das  Yerhalten 
der  Tuberkel-bacillen  im  Auswurf  wahrend  des 
Verlaufs  der  Lungenschwindsucht,"  Bed.  klin. 
Wochenschr.,  1882,  No.  45,  p.  679. 

BAUMGARTEN  (P.)  :  I.  "  Tuberkelbakterien," 
Centralbl.  f.  med.  Wissensch.,  1882,  No.  15,  19, 
Deutsche  med.  Wochenschr.,  1882,  p.  238;  II. 
"  Beitrage  zur  Darstellungsmethode  der  Tuberkel- 
bacillen."  Zeitschr.  f.  wissensch.  Mikroscopie, 
Bd.  L,  Hft,  1,  p.  51  ;  III.  "tleber  ein  bequems 
Verfahren  Tuberkel-bacillen  in  Sputis  nachzuwei- 
sen,"  Ctblt.  f.  d.  med.  Wiss.,  1882,  No.  25. 
IV.  "Ueber  das  Verhaltniss  von  Perlsucht  und 
Tuberculose."  Berl.  klin.  Wochenschr.  1880. 
Y.  "  Ueber  ein  neues  Reinculturferfahren  der  Tu- 


182  THE  TECHNOLOGY   OF 

berkelbacillen."  Ctbl.  f.  d.  med.  wiss.  1884, 
No.  22. 

BOLLINGER  (O.) :  "  Zur  Aetiologie  der  Tubercu- 
lose,"  Miinchen  (Rieger'sch  Univ.  Buchhdl.), 
1883.  II.  "  Ueber  Tuberkelbacillen  in  Eiter  einer 
tuberculosen  Kuh  und  iiber  Virulenz  des  Secretes 
einer  derartig  erkrankten  Milchdriisen."  Bayer- 
isches  arztl.  Int.  Bl.  1883.  No.  16. 

BOULEY  (H.)  :  "La  nature  vivante  de  la  conta- 
gion, contagiosite  de  la  tuberculose,"  Paris,  1884. 

BRUN  (J.)  :  "Note  sur  les  meilleurs  proceedes 
pour  reconnaitre  et  faire  des  preparations  mi- 
croscopiques  des  bactdries  de  la  tuberculose." 
Journal  de  Micrographie,  1882,  Vol.  VI.,  p.  500. 

BURRILL  (T.  J.)  :  "To  stain  Bacillus  tubercu- 
losis;" The  Microscope,  Vol.  IV.,  1884,  p.  6; 
Journal  de  Microgr.,  t.  VIII.,  1884,  No.  4,  p.  240. 

BURNET  (D.)  :  "Sur  la  tuberculose  experi- 
mentale."  Compt.  Kend.  T.  93  (1881),  p.  447, 
448. 

CELLI  (A.)  ET  GUARNIERI  (G.)  :  I.  "  Sopra 
talune  forme  crystalline  che  potrebbero  simulare 
il  bacillo  del  tuberculo"  (On  such  crystalline 
forms  as  maybe  mistaken  for  tuberculosis  bacilli), 
Acad.  dei  Lincei,  17,  gingno,  1883.  —  The  Micro- 
scope, Vol.  IV.,  1883,  No.  6,  p.  135.  — Zeitschr. 
f.  wiss.  Mikros.,  Bd.  1,  1884,  p.  590;  II.  "In- 
torno  alia  profilossi  del  Tuberculosi,  studi  d'igiene 
sperirnentale  "  (on  the  prophylaxis  of  tuberculosis, 


BACTERIA    INVESTIGATION.  183 

studies  in  experimental  hygiene),  Arch.  per.  Ic. 
se.  med.,  Vol.  VII.,  fasc.  3,  1884,  p.  233,  3  PI. 

CHYNE  (WATSON)  :  (On  Tuberculosis  bacilli). 
Practitioner,  April,  1883. 

CLARK  (J.  W.) :  "Preliminary  note  on  Bacillus 
tuberculosis,  Koch."  Nature,  Vol.  XXVII., 
p.  492. 

COLIN  (G.)  :  "  Sur  la  transmission  de  la  tuber- 
culose  aux  grandes  ruminants,"  Compt.  Rend.  T. 
99,  p.  1057  (Dec.  15,  1884). 

COPPOCK  :  "  On  Bacillus  tuberculosis,"  Microsc, 
News,  Vol.  III.,  1883,  No.  28,  p.  121. 

COZE  ET  SIMON  :  "  Recherches  de  pathologie  et 
de  therapeutique  experimentales  sur  la  tubercu- 
lose,"  Journ.  de  Micrographie,  t.  VIII.,  1884,  No. 
4,  p.  235. 

DEJ&RINE  ( J. )  :  "  Recherche  des  bacilles  dans  la 
tuberculose  calcifiee  et  caseo-calcifiee."  Revue  de 
Med.,  1884,  No.  12. 

DEMME  (R.)  :  "  Zur  diagnostischen  Bedeutung 
der  Tuberkelbacillen  fur  des  Kiridesalter."  Ber- 
liner klin.  Wochenschr.  1883,  No.  13. 

DETTWEILER  AND  MEISSEN  :  "  Der  Tuberkel- 
bacillus  und  die  chronische  Lungenschwindsucht, 
Berliner  klin.  Wochenschr.,  1883,  No.  4,  p.  97, 
also  No.  8,  p.  117. 

DOUTRELEPONT  i  "  Tuberkelbacillen  im  Lupus." 
Monats.  f.  prakt.  Dermat.  II.  No.  6. 

ERLICH  :  "  Eine  neue  Methode  der  Farbung  von 


184  THE   TECHNOLOGY   OF 

Tuberkelbacillen,"  Gesellschaft  der  Charite*-Aerzte 
in  Berlin.  Sitzung  von  27  April,  1882,  also  Berl. 
klin.  Wochenschr.,  Jan.,  1883,  p.  13,  also  Deutsch 
med.  Wochenschr.,  1882,  p.  267. 

EKMENGEN  :  "Le  microbe  de  la  Tuberculose," 
Revue  Mycologique  IV.,  No.  16.  II.  "Prepara- 
tion des  Bacteries  de  la  tuberculose.  Perfectionne- 
ments  apportes  a  la  methode  de  double  colora- 
tion." Journal  de  Micrographie,  1882,  p.  466. 

ERNST:  A: tides  in  two  August  numbers  of 
Boston  Med.  and  'Surg.  Journ.,  1883,  gives  exten- 
sive resume  of  the  literature  pertaining  to  Koch's 
discovery  of  Bacilli  of  Tuberculosis. 

ESCHLE  :  "  Tuberkelbacillen  in  dern  Ausflusse 
bei  mittelohr-Eiterung  von  Phthisikern."  Deutsch. 
med.  Wochenschr.  1883.  No.  30. 

FORMAD  (H.  F.)  :  "  The  Bacillus  of  Tubercule," 
New  York  Med.  Journ.,  1884,  Feb.  16,  also 
Amer.  M.  Microsc.  Journ.,  Vol.  V.,  No.  4,  1884. 

FRAENKEL  (B.)  :  I.  "Ueber  die  Farbung  des 
Koch'schen  Bacillus  und  seine  semiotische  Bedeu- 
tung  fur  die  Krankheiten  des  Respirationsorgane," 
Bed.  klin.  Wochenschr.,  1884,  No.  13;  II.  "Zur 
diagnose  des  tuberculosen  Kehlkopfgeschwurs " 
Ibid.  1883,  No.  4,  p.  58. 

FRANTZEL  (O.)  :  "Wie  weit  konnen  wir  das 
Nachweis  von  Tuberclebacillen  bis  jetzt  praktische 
verwerthen?"  Dtsch.  militairarztl.  Zeitschr.,  1883, 
Aug. 


BACTERIA    INVESTIGATION.  185 

GAFFKY  :  "  Ein  Beitrag  zum  Verhalten  der 
Tuberkelhacillen  im  sputum,"  Mitt,  aus  d.  kais. 
Gesundheitsarnt,  Bd.  2,  1884.  p.  126. 

GIACOMI  (DE)  (on  staining)  :  Fortschritte  der 
Med.,  1883. 

GIBBES  (H.)  :  I.  "On  a  rapid  method  of  dem- 
onstrating the  tubercle  bacillus  without  the  use  of 
nitric  acid,"  The  Lancet,  Vol.  L,  1883,  p.  771, 
Journ.  Roy.  Mic.  Soc.  Ser.  II.,  Vol.  III.,  1883, 
pt.  5,  p.  764,  Microsc.  News,  Vol.  III.,  1883,  No. 
33,  p.  248.  II.  "  A  new  method  for  the  detection 
of  the  tubercle  bacillus,"  British  Med.  Journ.,  14, 
Oct.,  1882. 

GIBOUX  :  "  Inoculabilitd  -de  la  tuberculose  par 
la  respiration  des  phthisiques."  Compt.  Rend.  T. 
94  (1882),  p.  1391. 

GROVE  (W.  B.)  :  A  synopsis  of  Bacteria  and 
yeast  fungi,  and  allied  species,  Schizomycetes  and 
Saccharomycetes.  Appendix  B.  On  the  stain- 
ing of  Bacillus  Tuberculosis.  Ehrlich's,  Gibbs's, 
and  Prideaux's  Methods.  87  figs.  8°.  London, 
1884. 

HARTZELL  (M.  B.)  :  "A  ready  method  for  the 
detection  of  the  Bacillus  tuberculosis,"  Med.  Times, 
Jan'y  26,  1884,  The  Microscope,  Vol.  IV.,  1884, 
No.  5,  p.  115,  Amer.  M.  Microsc.  Journ.,  Vol. 
IV.,  1884,  No.  4,  p.  76,  Journ.  Roy.  Mic.  Soc., 
Ser.  II.,  Vol.  IV.,  1884,  pt.  4,  p.  653. 

HERON  (G.  A.):    "Ehrlich's   method   for  the 


186  THE  TECHNOLOGY   OF 

detection  of  Tubercle  Bacilli  in  sputum."  Brit. 
Med.  Journ.,  No.  1137  (1882),  p.  735. 

HILLER  :  "  Ueber  initiale  Hamoptce  und  ihre 
Beziehung  zur  Tuberculose."  Dtsch.  ined.  Wo- 
chenschr., No.  47,  1882. 

IRSAI  (A.)  :  "  Zur  Diagnostik  der  Tuberculose 
des  Harn-apparates  auf  Gruncl  des  Befundes  von 
Koch'schen  Tuberkelbacillen  im  Hani."  Wiener 
med.  Presse,  1884,  Nos.  36,  37. 

KAATZER  (P.):  "Die  Technik  der  Sputum 
Untersuchung  auf  Tuberkel-Bacillen,"  2d  Aufl., 
Wiesbaden,  1884. 

KAROP  (G.  C.)  :  "On  a  specimen  of  Bacillus 
tuberculosis  prepared  by  Dr.  Gibbs  method," 
Journ.  Quek.  Microsc.  Club,  Vol.  I.,  1883, 
No.  1. 

KLEBS  (E.)  :  "Ueber  Tuberculose,"  Prager 
med.  Wochenschr.,  1877,  Nos.  42  and  43.  (Tbe 
first  to  propose  the  theory  that  the  tuberculosis 
virus  must  contain  Bacteria,  cultivated  these  in 
white  of  egg.) 

KOCH  (R.)  :  I.  "Die  Aetiologie  der  Tu- 
berculose," Verhandl.  des  Congresses  fur  innere 
Medicin,  1882;  II.  "Die  Aetiologie  der  Tu- 
berculose," Berl.  klin.  Wochenschr.,  1882,  p. 
221,  also  Deutsch  med.  Wochenschr.,  1882, 
Nov.  15,  16,  18;  III.  Same  title.  Mithei- 
lung  aus  der  kais.  Gesundheitsamt,  Bd.  2, 
1884. 


BACTERIA    INVESTIGATION.  187 

KOENIG  (FR.)  :  "Die  Tuberculose  der  Knochen 
und  Gelenke,  auf  Grund  eigener  Beobachtungen," 
gr.  8°.  1883. 

KORAB  :  "  Influence  of  Helenin  on  Bacillus  Tu- 
berculosis," Berlin  med.  Central-Zeitung,  1882, 
Nos.  30,  31. 

KREDEL  :  "  Klinische  Erfahrungen  iiber  Tu- 
berkelbacillen."  Bericht  der  Oberhessischen 
Gesellschaft  fiir  Natur.  u.  Heilkunde.  Giessen, 
1883, 

KUSSNER  :  "  Beitrag  zur  Impftuberculose." 
Deutsche  med.  Wochenschr,  1883.  No.  36. 

LACHMANN  :  "  Zur  Kenntniss  der  Tuberkel- 
bacilen."  Dtsch.  med.  Wochenschr.,  1884,  No. 
13. 

LENZ,  V.  :  "  Experimentelle  Untersuchungen 
iiber  die  Infectiossitat  des  Blutes  und  Urines  Tu- 
berculoser."  Dissert.  Berlin,  July,  1881. 

LEYDEN  (E.)  :  "  Klinisches  iiber  den  Tu- 
berkel bacillus."  Zeitschr.  f.  klin.  Med.,  VIII., 
p.  375.  —  Ctbl.  f.  d.  med.  Wiss.,  1885,  p. 
152. 

LICHTHEIM  (L.)  :  "Zur  Diagnostischen  Ver- 
werthung  der  Tuberkelbacillen."  Fortschritte  der 
Medicin,  Bd.  1,  Hft.  1,  p.  4,  1883. 

LONG:  (staining  method).  Berliner  klin. 
Wochenschr.,  January,  1883,  p.  33  (vid.  Pfeiffer, 
below) . 

MALASSEZ  (L.)  ET  VIGNAL  (W.)  :  "Sur  le  Mi- 


188  THE  TECHNOLOGY  OF 

cro-organisme  de  la  Tuberculose  Zooglseique." l 
Compt.  Rend.,  T.  98,  July  28,  1884,  p.  203. 
Also  5  Nov.,  1883.  Soc.  de  Biologie,  Seances, 
12-19  Mai  et  9  Juin,  1883.  Archiv  de  Phys.,  15 
Nov.,  1883. 

MENSCHE  (On  Staining)  :  "  Yortrag,  gehalten 
in  der  med.  Sect,  des  Niedenh."  Yereins  fur 
Natur.  u.  Heilk.  zu  Bonn,  1883. 

MULLER  (F.)  :  "Ueber  die  Diagnostiche  Be- 
deutung  der  Tuberkelbacillen "  (Yerhand'l.  phys. 
med.  Gesellsch.  zu  Wiirzburg,  1883,  pp.  7,  8°. 

NATHAN,  J.  S.  :  "Ueber  das  Yorkormnen  von 
Tuberkelbacillen  bei  Ottorhoen"  (Aus  d.  med. 
klin.  Institut  zu  Miinchen.  Otiatrisches  Ambu- 
latorium  von  Dr.  Bezold).  Deutsches  Archiv  f. 
klin.  Med.  xxxv.  p.  491. 

NEGRI  (A.  F.) :  Coloration  des  spores  dans 
bacilles  de  la  tuberculose.  Journal  de  Micro- 
graphie,  T.  viii.,  1884,  No.  6,  p.  349. 

ORTH,  J.  :  "Notizen  zur  Farbetechnik."  Berl. 
klin.  Wochenschr.,  1883,  No.  28,  p.  421. 

PETERS  :  "  Nachvveis  der  Tuberkelbacillen  in 
Schnitten  durch  die  Doppel-farbung ;  '  Gentiana- 


1  "  Nous  avons  appele  tuberculose  zooglwique  une  affection 
caus^e  par  1'inoculation  de  produits  tuberculeux,  dans  lesquels 
nous  n'avions  pas  trouve  de  bacilles  (tubercule  cutane'  parvi  d'abces 
ossifluent)  ayant  tous  les  caracteres  cliniques  et  anatomo-patholo- 
giques  de  certaiiies  tuberculoses,  mais  pre'sentant,  pendant  les  pre- 
mieres ge"ne'rations  tout  an  moins  des  amas  zoogloeiques  de  micro- 
cocques  et  pas  de  bacilles."  1.  c.  p.  494. 


BACTERIA    INVESTIGATION.  189 

violet.'  Anilingelb  ohne  Salpetersaure  entfar- 
bung."  Berl.  klin.  Wochenschr.,  1883,  No.  24, 
26,  p.  365. 

PETRI  :  "  Zur  Farbung  des  Koch'schen  Bacillus 
in  Sputis  sowie  iiber  das  gleiche  Verhalten  ein- 
iger  Pilzzellen."  Berl.  klin.  Wochenschr.,  No.  26, 
p.  739,  1883. 

PFEIFFER  (Aug.)  :  "  Ueber  die  Regelmassigkeit 
des  Vorkommens  von  Tuberkelbacillen  im  Auswurf 
Schwindsuchtiger."  Berl.  klin.  Wochenschr., 
1883,  Jan'y,  p.  32  (gives  Long's  method). 

PFUHL-PETRI  (Staining  method)  :  Deutsche 
militararztliche  Zeitschrift,  1884,  Heft.  3. 

PRIOR  :  "  Beitrag  zur  Farbarkeit  des  Tuberkel- 
bacillus."  Berl.  klin.  Wochenschr.,  1883,  No.  33, 
p.  497. 

PUTZ  :  "  Ueber  d.  Beziehungen  der  Tuberculose 
des  Menschen  zur  Tuberkulose  des  Thiere."  Stutt- 
gart, 1883. 

"  Photographing  Bacillus  tuberculosis."  Journ. 
Eoy.  Micro.  Soc.,  Ser.  II.,  Vol.  IV.,  1884,  pt.  4, 
p.  627. 

QUINLAN  (J  B.)  :  "Bacillus  Mounting."  The 
Microscope,  Vol.  III.,  1883,  p.  138.— Med.  and 
Surg.  Reporter,  1883.  Journal  de  Micrographie, 
1883,  p.  441. 

RANSOME  (A.)  :  "  Bacilli  in  condensed  aqueous 
vapour  of  the  breath  of  phthisical  persons."  Proc. 
Roy.  Soc.  XXXIV.,  1882,  p.  274-5. 


190  THE   TECHNOLOGY  OF 

RAYMOND  ET  ARNAUD  :  "  Recherches  Experi- 
mentales  sur  1'^tiologie  de  la  Tuberculose." 
Extr.  des  Archives  G£ner.  d.  M<3d.,  1883,  Jan'y. 

REICHENBACH  (H.)  :  "Die  Entdeckung  der  Tu- 
berculose Bacillen  durch  Dr.  Robert  Koch." 
Humboldt,  Monatsschrift  fur  die  gesammten  Na- 
turwissenschaften.  Hft.  VII.,  1882. 

REINSTADLEK  (F.  A.):  "  Ueber  Impftubercu- 
lose."  Arch.  f.  exp.  Pathol.  u.  Pharm.  Bd.  XI. 
1879,  p.  103-121. 

RINDFLEISH  :  I.  "  Ueber  die  Methode  der  Bacil- 
len farbung  in  Sputum."  Berl.  klin.  Wochenschr. 
No.  12,  1883,  p.  183.  II.  "Demonstration  von 
Tuberkelbacillen."  Sitzber.  d.  phys.  med.  Gesell. 
zu.  Wurzburg,  Jahrgang,  1882,  p.  22. 

ROSENSTEIN  (S.)  :  "  Vorkommen  der  Tuberkel- 
bacillen im  Harn."  Ctbl.  f.  d.  med.  Wiss.  1883, 
No.  5. 

RUHLE  AND  LICHTHEIM  :  "  Einfluss  der  Ent- 
deckung der  Bacillen  auf  die  Pathologie,  Proplry- 
laxe  und  Therapie  der  Tuberculose."  Vorhand- 
lung  des  Congresses  fur  innere  Med.  1882,  II. 
Congress. 

SALOMONSEN  :  "  Om  Indpodning  af  Tuber- 
kulose,  sarligt  i  Kaninens  Iris."  Nordiskt  Med- 
icinskt  Arkiv,  1879,  Bd.  XV.,  No.  12-19. 

SAUVAGE  :  "  De  la  valeur  diagnostique  de  la 
presence  des  bacilles  de  Koch  dans  les  crachats." 
Paris  (Delahaye  et  Leer.),  1884. 


BACTEKIA    INVESTIGATION.  191 

SCHLEGTENDAL  :  "  On  Schizomycetes  in  Tuber- 
cular Abscesses."  Fortschritte  der  Medicin,  p. 
537,  1883. 

SCHMIDT  (H.  D.)  :  (Bacillus  Tuberculosis.) 
Louisville  Med.  Herald,  IV.  1883,  pp.  459-76,  6 
figs.  Considers  it  simply  a  fat  crystal. 

SCHILL  :  "  Ueber  den  Nachweis  von  Tuberkel- 
bacillen  im  Sputum."  Deutsch.  med.  Wochenschr, 
1883,  No.  2. 

SCHUCHARDT  UND  KuAUSE  :  "  Ueber  das  Vor- 
kommen  der  Tuberkelbacillen  bei  fungosen  und 
scrophulosen  Entzundungen."  Fortschritte  der 
Medicin.,  No.  9,  1883. 

SENKEWITSCH  (Staining  method) :  Revue  fiir 
Thierheilkunde,  Bd.  7,  No.  7,  1884. 

SMITH  (T.):  "Method  of  demonstrating  the 
presence  of  the  Tubercle  Bacillus  in  Sputum." 
American  Monthly  Micros.  Journ.,  1884,  p. 
196-9. 

SPINA  :  "  Studien  iiber  Tuberculose  (Wien 
1883)  und  deren  Enwiderung  durch  Koch  und 
Ehrlich."  Bericht  iiber  die  Sitzung  des  Yereins 
f.  innere  Medicin,  5  Marz,  1883. 

SOMARI  E  BRUGNATELLI  :  "  Studi  spermentali 
sul  bacillo  della  tuberculosi  (Experimental  studies 
on  the  Bacilli  of  Tuberculosis)."  Redii  R.  Instit. 
Lombardo,  Yol.  XYI.,  1883,  No.  16. 

STERNBERG  (G.  N.)  :  "-Etiology  of  Tuberculo- 
sis." Abstract  of  a  paper  read  before  Sect.  F. 


192  THE  TECHNOLOGY   OF 

(Biology)  of  the  Amer.  Assoc.  Adv.  Sci.  Phila- 
delphia, Sept.  9,  1884. 

STOWELL  (C.  H.)  :  "Bacillus  staining."  The 
Microscope,  Vol.  IV.  1884,  No.  4,  p.  79. 

TAPPEINER  :  "  Zur  Frage  der  Constagiositat  der 
Tuberculose,  Experimentelle  Untersuchungen." 
Deutsche  Arch.  f.  klin.  Med.,  Bd.  XXIX.,  p. 
595-600. 

TOUSSAINT  :  "  Sur  le  parasitisme  de  la  tubercu- 
lose."  Compt.  Rend.  T.  93  (1881),  p.  350-3. 

VERAGUTH  (C.):  "Ueber  den  Nachweis  der 
Tuberkelbacillen  in  Chromsaure  Praparaten."  Berl. 
klin.  Wochenschr.  No.  13,  1883,  p.  190. 

WEIGERT  (Staining  method  for  tubercle  bacilli)  : 
"  Farbungs  Methoden,"  by  Dr.  Hugo  Plaut,  Leip- 
zig, 1885,  p.  19. 

WEICHSELBAUM  (A.)  :  "Ueber  Tuberkelbacillen 
in  Blute  bei  allgemeiner  acuter  Tuberculose." 
Wien  med.  Wochenschr.,  1884,  No.  12,  13.  vid. 
also  vol.  for  1883.  II.  (Inhalation  of  tubercular 
matter).  Wiener  med.  Jahrb.  1883. 

WESNER  (F.)  :  "Ueber  das  Vorkommen  derTu- 
berkel  bacillen  in  der  Organen  Tuberkuloses." 
Deutsches  Archiv.  f.  klin.  Med.  XXXIV.,  p. 
583. 

WILLIAMS  (Th.) :  "  On  the  relations  of  the 
Tubercle  Bacillus  to  Phthisis."  Lancet,  1883,  No. 
3127. 

WILLIAMS  (C.  T.)  :  "  Influence  of  Culture  fluids 


BACTERIA    INVESTIGATION.  193 

and  Medicinal  reagents  on  the  Growth  and  Devel- 
opment of  Bacillus  Tuberculosis.7'  Journ.  Roy. 
Mic.  Soc.,  1884,  p.  932. 

ZIEHL  (Fr.):  I.  "Zur  Farbung  des  Tuberkel- 
bacillus."  Deutsch  med.  Wochenschr.  1882,  No. 
33,  p.  451.  II.  Zur  Lehre  von  der  Tuberkel- 
bacillen,  inbesonderes  iiber  deren  Bedeutung 
fiir  Diagnose  und  Prognose."  Ibid.,  1883, 
No.  5. 

Luc  (H.)  :  "De  la  tuberculose  de  la  con- 
jonctive  compared  au  lupus  de  cette  muqueuse, 
contribution  a  la  differenciation  clinique  de  ces 
deux  affections."  8°.,  39  pp.,  Paris  (Davy), 
1883. 

PFEIFFER  (A.)  :  "  Tuberkelbacillen  in  der  Lu- 
pus erkrankten  Conjunctiva."  Berl.  klin.  Woch- 
enschr., July,  1883,  p.  431 

PAGENSTECHER  UND  PFEIFFER  :  "  Lupus  oder 
Tuberculose."  Berl.  klin.  Wochenschr.  1883, 
No.  19,  p.  282. 

SCHULLER  (M.)  :  "  Histologische  Studien  iiber 
Mikrokokken  des  Lupus."  Ctbl.  f.  Chir.,  1881, 
No.  46. 

TYPHOID  FEVER. 

Bacillus  typhi-abdominalis  ( Brautlecht ) . 

The  Bacilli  of  typhoid  fever  are  decolorized  when 

prepared    according   to    Gram's   general   method. 

Klebs  employed  haematoxylin  as  a  staining  agent 


194  THE  TECHNOLOGY   OF 

in  his  researches.  There  is  a  difference  of  opin- 
ion as  to  the  specific  microbe  of  typhoid  fever 
being  a  Bacillus,  —  according  to  Letzerich  it 
is  a  Micrococcus,  which  he  demonstrated  as  fol- 
lows :  — 

Letzerictis  Method.  —  He  employed  partly  fresh 
and  partly  hardened  organs,  made  sections,  and 
cleared  them  up  in  weak  caustic  potash  solution  or 
a  solution  of  carbonate  of  soda,  or,  better  still,  in 
highly  diluted  glacial  acetic  acid  (1  :  3),  as  this 
does  not  destroy  the  nuclei  and  allows  the  relation 
of  the  Schizomycetes  to  the  tissue  to  be  seen.  In 
sections  of  intestine  hardened  in  alcohol,  he  em- 
ployed the  acid  one  part  to  two  of  water,  and 
allowed  them  to  remain  in  it  for  half  an  hour, 
added  a  little  glycerine,  and  mounted  on  a  slide ; 
this  gave  a  beautiful  preparation,  in  which  both 
nuclei  and  microbes  were  apparent. 

Rindfleisch's  Method.  —  ( 1 . )  He  took  water  from 
a  suspected  well,  allowed  a  drop  to  dry  upon  a 
slide,  colored  it  with  a  weak  methyl-violet  solu- 
tion, washed  it  a  moment  in  water,  dried  it,  and 
mounted  in  Canada  balsam.  Eesult :  numerous 
deeply  blue  colored  rod  Bacteria. 

(2.)  Inoculated  with  the  water  a  culture  of 
human-flesh  gelatine,  using  all  precautions  against 
external  contamination.  Result :  a  rapid  solution 
of  gelatine  by  the  culture. 


BACTERIA  INVESTIGATION.  195 


LITERATURE. 

BOENS  :  "La  fievre  typhoide,  ses  causes,  son 
traitemdht  et  sa  prophylaxie."  A  cad.  roy.  de  med. 
deBelgique.  Bulletin,  1883,  3dser.  T.  XYII.,  p. 
176. 

BRAUTLECHT  :  "  Typhus  Bakterien  und  Trink- 
wasser."  Virch.  Archiv,  1880,  p.  80. 

EBERTH  :  "  Neue  Untersuchungen  iiber  d.  Bacil- 
lus d.  abdominal  Typhus."  Archiv  f.  path.  Anat. 
u.  Pkysiol.  u.  f.  klin.  Med.  Bd.  83  (1881).  II. 
"Die  Organismen  in  d.  Organen  bei  Typhus  ab- 
dominalis."  Virchow's  Archiv  Bd.  81,  1880,  p. 
58,  vid.  also  Bd.  87. 

FRANK  (A.)  :  "  Zur  Aetiologie  des  abdominal 
Typhus."  Bayr.  artz.  Int.  BL,  1881,  No.  23. 

FRIEDLAENDER  :  "  Bacillus  des  abdominal  Ty- 
phus." Sitzung  d.  Ver.  f.  innere  Medecin,  Ber- 
lin, 1881,  17  Nov. 

GAFFKY  :  "  Zur  Aetiologie  des  abdominal  Ty- 
phus." Mittheilung  aus  d.  kais  Reichsgesund- 
heitsamt,  Bd.  2,  1884. 

HANOT  (V.)  :  "  Miliaire  bacteridienne  dans  la 
fievre  typhoide."  Rev.  d.  Med.,  1881,  10  Oct. 

HEIN  (I.)  :  '  Typhusbacillen  im  Milzblute 
resp.  Milzsafte."  Ctbl.  f.  d.  Med.  Wiss.  1884, 
No.  40. 

RLEBS  (Eo.)  :  I.  "Der  Ileotyphus  eine  Schizo- 
mycose."  Archiv  f.  exp.  Pathol.  u.  Pharrn.  Bd. 


196  THE  TECHNOLOGY  OF 

XII.  Heft.  3,  p.  231  (1880).  II.  "Bacillus  des 
Abdominal  typhus  und  der  typhose  Process." 
Ibid.  Bd.  XIII.,  p.  381.  Taf.  IV.,  V.,  VI. 

KLEIN  (E.)  :  (On  Typhoid  Fever).  Keports  of 
the  Medical  Officer  of  the  Privy  Council,  1875. 

KOCH  (R.)  :  (Typhoid  Fever).  Mittheilungen 
a.  d.  k.  Gesundheitsamt,  Bd.  1,  1881. 

LETZERICH  (L.)  :  I.  "  Studien  iiber  Typhus  ab- 
dominalis."  Yirchow's  Archiv,  Bd.  68.  II.  "Ex- 
perimentelle  Untersuchungen  iiber  Typhus  abdom- 
inalis."  Archiv  f.  experim.  Pathol.  u.  Pharm. 
Bd.  9,  p.  312. 

LUDWIG  (E.)  :  "Beitrag  zur  Frage  der  Entsteh- 
ung  und  Yerbreitung  des  Abdominaltyphus." 
Wiirtemberger  med.  Corr.  Bl.  1882,  No.  5-6. 

MEYEK  (Wn.)  :  "  Untersuchungen  iiber  den 
Bacillus  des  abdominal  Typhus."  (Aus  dem  stadt. 
allg.  Krankenhaus  zu  Berlin.)  Dissert.,  Berlin, 
Sept.,  1881. 

RAPPIN  :  "  Des  bacte*ries  de  la  bouche  a  1'etat 
normale  et  dans  la  fievre  typhoide."  Paris, 
1881. 

RINDFLEISH  :  "Ueber  Trinkwassertyphus."  Sitz- 
ungsber.  der  phys.  med.  Gesellsch.  zu  Wiirzburg, 
Jahrgang  1882,  p.  133. 

ROTH  (F.)  :  "  Ueber  die  Yerbreitung  des  Ty- 
phoides  (abdominal  Typhus)  nach  Wasserlaufer." 
Bayr.  arztl.  Int.  Bl.,  1881,  No.  44. 

TAYON  :  "  Sur  le  microbe  de  la  fievre  typhoide 


BACTERIA  INVESTIGATION.  197 

de  I'homme ;  culture  et  inoculations."  Compt. 
Rend.  T.  99,  p.  331,  Aug.  18,  1884. 

TIZZONI  :  "  Studi  sulla  natura  del  tifo  abdomi- 
nale."  Annali  universale  di  Medicina,  Vol.  251, 
1880. 

WERNICH  (A.):  "Typhus  Bacillen."  Arztl. 
Int.  Bl.  1881,  No.  44.  if"  Typhus  Bacillen."  Sitz- 
ung  des  Yereins  fiir  innere  Med.,  Berlin,  1880,  4 
July.  II.  "  Studien  und  Erfahrungen  iiber  den 
Typhus  abdorainalis."  Zeitschr.  f.  klin.  Med.  Bd. 
IY.,  H.  1.  III.  "  Der  abdominal  Typhus,  Unter- 
suchungen  iiber  sein  Wesen,  seine  Todlichkeit  und 
seine  Bekampfung"  1882,  Berlin  (A.  Hirschwald). 

WHOOPING  COUGH. 

Burger's  Method.  —  Cover-glass  preparations 
made  according  to  general  rules,  and  stained  with 
aqueous  solutions  of  fuchsin  and  methyl-violet. 

LITER  ATUKE. 

BURGER  (CARL)  :  "  Der  Keuchhustenpilz."  Ber- 
lin, klin.  Wochenschr.,  Jan.,  1883,  p.  7. 

CONCEPTIONS  OF  THE  LACHRYMAL  DUCTS. 

Cladothrix  foersteri  (Colin). 

No  special  methods,  so  far  as  I  can  ascertain, 
have  been  given  for  the  study  or  preparation  of  the 
Bacteria  found  in  the  concretions  of  the  lachrymal 
ducts. 


198  THE  TECHNOLOGY   OF 

LITERATURE. 

COHN  (F.)  :  Beitr.  z.  Biol.  d.  Pflanzen.  Vol. 
1,  p.  186. 

FOERSTER  :  "  Pilzmasse  in  unteren  Thranen-Ka- 
nalchen."  Arch.  f.  Opthalni.  XV.,  1. 

GOLDZIEHER  (W.)  ;  "  Streptothrix  Foersteri  im 
unteren  Thranenrohrchen."  Ctbl.  f.  prakt.  Au- 
genheilk.  1884,  Febr. 

GRAEFE  :  "  Ueber  Leptothrix  in  d.  Thranen- 
rohrchen." Archiv  f.  Opthalm.  Bd.  XVI.,  1. 

REUSS  (A.  v.)  :  ft  Pilzkonkretionen  in  den  Thra- 
nenrohrchen." Wien  med.  Pr.,  1884,  No.  7  u.  8. 

DENTAL  CAEIES. 
Leptothrix  buccalis  (Kobin)  etc. 

Prof.  Ferd.  Cohn  l  calls  attention  to  the  fact  that 
in  a  letter  dated  September  14, 1683,  A.  Van  Leeu- 
wenhoek  gave  notice  to  the  Royal  Society  that 
with  the  aid  of  his  microscope  he  had  discovered  in 
the  white  substance  adhering  to  his  teeth  very 
little  animals  moving  in  a  very  lively  manner. 
They  were  the  first  Bacteria  the  human  eye  ever 
saw. 

Leber's  Method.  —  If  the  Leptothrix  fibres  are 
found  in  an  acid  medium,  it  is  sufficient  to  add 
iodine  to  stain  the  contents  blue  or  violet.  If  they 

i  Nature,  xxix.,  1883,  p.  154. 


BACTERIA    INVESTIGATION.  199 

occur  in  an  alkaline  medium,  acidulate  with  dilute 
hydrochloric  or  acetic  acid,  then  add  iodine.  Pre- 
pare cover-glasses  according  to  general  directions. 
Miller's  Method.  —  Dr.  Miller  made  one  thousand 
sections  of  carious  teeth,  of  which  not  one  failed 
to  show  Bacteria  deep  in  the  tooth  tissue,  whether 
the  tooth  was  a  living  or  dead  one.  The  contrary 
results  obtained  by  former  observers  are  due,  ac- 
cording to  Miller,  to  their  methods  of  coloring, 
which  were  not  suited  to  the  case  in  hand.  To 
show  the  grouping  of  the  Bacteria  heaps,  whether 
in  transverse  or  longitudinal  sections,  use  an  alco- 
holic solution  of  Magdala  red.  Nearly  as  good 
results  are  given  with  fuchsin,  methyl-blue,  or 
Bismarck-bro  w  n . 

LITERATURE. 

ARNDT  :  "  Beobacht.  an  Spirochaete  denticola." 
Archiv  f.  path.  Anat.,  Physiol.  u.  klinisch.  Med., 
1880,  Bd.  79. 

BATJME  :  "  Odontologische  Forschungen."     II. 

LEBER  UND  ROTTENSTELN  :  "  Untersuchungen 
iiber  Caries  der  Zahne."  Berlin,  1867. 

MILLER  (W.  D.)  :  I.  "  Der  Einfluss  der  Micro- 
organismen  auf  die  Caries  der  menschlicher  Zahne.'' 
Archiv  f.  experim.  Pathol.  u.  Pharm.  XVL,  1882. 
II.  "  Ueber  der  Caries  der  Zahne."  Corresp.  f. 
Zahnarzte.  Bd.  XIII.  III.  "  Ueber  einen  Zahn- 
spaltpilze,  Leptothrix  gigantse."  Ber  d.  deutsch. 


200  THE  TECHNOLOGY  OF 

bot.  Gesellsch.  1883,  H.  5.  IV.  "  Zur  Kenntniss 
der  Bakterien  in  der  Mundhohle."  Deutsche  med. 
Wochenschr.  27  Nov.  1884.  No.  48,  p.  781. 

AREA  CELSII. 
(Alopecia  areata.) 

Buchner's  Method  was  to  remove  a  diseased  hair 
with  sterilized  pincers,  and  place  it  entire  into 
ordinary  culture  fluids. 

Von  Sehlerfs  Method  for  staining  the  micrococci 
of  this  disease  is  to  place  the  hairs  in  toto,  after 
having  removed  all  oil  and  fat  by  means  of  chloro- 
form and  ether,  in  a  very  dilute  anilin-oil-fuchsin 
solution,  or  a  carbolic  fuchsin  solution  for  twenty- 
four  hours,  then  wash  in  hydrochloric  acid  alcohol, 
and  remove  the  acid  with  distilled  water.  Give 
double-staining  with  methyl-blue  or  gentian- 
violet  solution,  wash  in  absolute  alcohol,  pass 
through  oil  of  cloves,  and  mount  in  Canada  bal- 
sam. Simple  staining  with  one  aniline  color 
leads  to  no  result,  because  the  hair  cells  color 
in  a  similar  manner.  For  transverse  sections, 
dehydrate  the  hairs  with  absolute  alcohol,  and 
allow  them  to  lie  for  some  time  in  chloroform, 
then  imbed  in  a  chloroform  paraffine  solution,  and 
cut  sections  0.01.  mm.  thick,  fasten  these  to  the 
slide  by  the  ordinary  methods,  e.  g.,  by  painting 
the  slide  over  with  a  mixture  of  oil  of  cloves  and 
collodion  before  placing  the  sections  upon  it,  and 


BACTERIA    INVESTIGATION.  201 

allowing  it  to  dry  before  proceeding  further) 
and  stain  with  a  strong  solution  of  aniline-oil- 
fuchsin,  or  of  carbolic  fuchsin,  then  wash  in  acid- 
ulated (HC1)  alcohol,  and  remove  the  acid  with 
distilled  water.  Give  second  staining  with  a  con- 
centrated aqueous  solution  of  gentian- violet,  wash 
in  absolute  alcohol,  pass  through  oil  of  cloves,  and 
mount  in  Canada  balsam. 

LITERATURE. 

BALZER  :  "  Contribution  a  Fetude  de  l'eryth£me 
tricophytique."  Archiv  de  Physiol.  3°  ser.  1883, 
T.  1,  p.  171. 

BUCHNER  (H.)  :  "  Kritische  Bemerkungen  zur 
Aetiologie  der  Area  Celsii."  Virchow's  Archiv, 
Bd.  74.  p.  527.  (1878). 

MICHELSON  (P.)  :  "Bemerkungzu  den  Arbeiten 
des  Herrn  Dr.  v.  Sehlen  iiber  die  Aetiologie  der 
Alopecia  areata  (area  celsii)."  Virchow's  Archiv, 
Bd.  99,  Hft.  3,  p.  572. 

YON  SEHLEN  (D.)  :  "  Mikrokokken  bei  Area 
Celsii."  Fortschritte  der  Med.  Bd.  1.  No.  23.  p. 
763.  (1883).  II.  "Zur  Aetiologie  der  Alopecia 
areata."  Virchow's  Archiv,  Bd.  99,  2  Hft.  (1885). 

CHICKEN  CHOLERA. 

(Micrococcus  gallicidus.) 

There  is  some  uncertainty  as  to  whether  the 
microbe  of  this  disease  is  a  dumb-bell  Mi- 


202    •  THE  TECHNOLOGY   OF 

crococcus  (Diplococcus) ,  or  a  Bacterium.  Klein 
intimates  that  Pasteur  used  impure  cultures, 
and  that  the  organism  is  probably  a  Bacterium 
termo. 

Barthelemy's  Method.  —  A  hen  died  of  this 
disease  after  having  laid  fourteen  eggs.  Barthel- 
emy  incubated  these  eggs,  marking  them  in  order 
to  distinguish  them  from  a  second  batch  used  as  a 
'  control '  experiment.  Underneath  the  shell  and 
on  the  surface  of  the  allantois  he  found  lacunae  of 
blood,  black,  and  having  a  special  odor  like  that 
of  fowls  dead  of  the  cholera.  This  blood  was 
filled  with  Bacteria,  while  the  amniotic  fluid  con- 
tained very  minute  monads.  It  is  evident  the 
egg  contained  the  germs  of  the  microbes  with 
which  the  liquids  of  the  mother  were  filled,  but 
these  did  not  develop  until  the  allantois  furnished 
oxygen. 

Pasteur's  Method.  —  Neutralized  urine  and  yeast 
water  were  first  tried  as  culture  fluids,  but  found 
unadapted  to  the  purpose.  A  meat  or  chicken 
broth  neutralized  with  carbonate  of  potash  and 
sterilized  at  a  temperature  of  100°-115°  C.,  was 
found  to  be  well  suited ;  the  Bacteria  developing 
rapidly  within  one  hour.  After  cultivating  the 
microbes  for  several  days  he  filtered  them  all  out 
and  inoculated  a  chicken  with  the  filtrate,  produc- 
ing the  disease  in  a  mild  form. 


BACTERIA    INVESTIGATION.  203 


LITERATURE. 

BABES  :  (On  chicken  cholera) .  Archiv  de  Phys- 
iol.  July,  1883,  p.  49. 

BARTHELEMY  (A.)  :  "De  1'incubation  des  ceufs 
d'une  poule  atteinte  du  cholera  des  poules." 
Compt.  Rend.  T.  96.  p.  1322. 

PASTEUR  (L.)  :  I.  "  Verbal  Observations  on 
Chicken  Cholera."  Compt.  Rend.  T.  79-89.  II. 
"  Behavior  of  Bacteria  of  Chicken  Cholera  to 
cold."  Ibid.  79.  No.  24.  III.  "  Sur  les  maladies 
virulente  et  en  particulier  sur  la  maladie  appelde 
vulgairement  cholera  des  poules."  Ibid.  T.  90. 
p.  239.  (1880).  German  translation  in  Arch.  f. 
experira.  Pathol.  Bd.  12,  Heft.  4,  p.  344.  IV. 
"  Sur  le  Cholera  des  poules  ;  etudes  des  conditions 
de  la  non-recidive  de  la  maladie  et  de  quelques 
autres  de  ses  caracters."  Compt.  Rend.  T.  90 
(1880).  p.  952  and  1030.  Compare  also  Transac- 
tions of  the  International  Medical  Congress  in 
London,  1881,  vol.  L,  p.  87. 

PERRONCITO  :  "  Ueber  das  epizootische  Typhoide 
der  Huhner."  Archiv  f.  wissenschaftl.  u  pract. 
Thierheilkunde,  1879,  p.  22. 

SEMMER  :  "  Huhnerpest."  Dtsch.  Zeitschrft.  f. 
thier.  Med.  u.  vergl.  Pathol.,  1878. 

TOUSSAINT  (H.)  :  "  Identite  de  la  septicdmie 
expe"rimentale  aigue  et  du  cholera  des  poules." 
Compt.  Rend.  T"  91,  p.  301  (1880). 


204  THE  TECHNOLOGY  OF 


DIPHTHERIA. 
Micrococci  diphtheriticus  (Colin. ) 

Both  Micrococci  and  Bacilli  occur  in  diptheritic 
membranes,  and  there  is  a  division  among  investi- 
gators as  to  which  is  the  vera  causa  morbi.  Buhl, 
Huter,  Formad,  Klein,  and  others  claim  Micro- 
cocci  to  be  the  active  agents;  while,  as  will  be 
seen  below,  Loeffler  considered  these  to  be  of 
only  secondary  importance.  Further  investiga- 
tions should  be  made  with  pure  cultures  and  inocu- 
lations from  these. 

Loeffler' s  Method.  —  On  account  of  the  great 
variety  of  Bacteria  inhabiting  the  mucous  mem- 
branes of  men,  Loeffler  desired  a  staining  fluid  by 
which  all  known  Bacteria  might  be  stained,  and 
used  as  such  the  following  :  — 

Add  to  30  cc.  of  concentrated  alcoholic  methyl- 
blue  solution  100  cc.  of  caustic  potash  solution  (1 
part  to  1,000  parts  of  water).  It  is  sufficient  for 
sections  to  allow  them  to  remain  only  a  few  mo- 
ments in  the  solution,  which  will  give  an  intense 
stain  to  all  known  Bacteria.  After  staining,  the 
sections  are  placed  for  a  short  time  in  a  half  per 
cent  acetic  acid  solution,  moved  about  for  several 
seconds,  and  then  dehydrated  in  absolute  alcohol, 
passed  through  oil  of  cedar,  and  mounted  in 
Canada  balsam.  By  this  process  Loeffler  found  two 
peculiar  microbes  constantly  present  in  diphtheria : 


BACTERIA    INVESTIGATION.  205 

(1)  A  chain-building  Micrococcus,  which  he  culti- 
vated according  to  general  rules,  in  stiff  blood- 
serum  and  on  cooked  potato,  at  ordinary  tem- 
'peratures  and  in  a  breeding  oven.  (2)  A  Ba- 
cillus like  that  discovered  by  Klebs  ("fur 
bacillar  Diphtheria");  this  flourished  best  at 
37°  C.  on  a  mixture  of  three  parts  of  calves' 
or  sheep's  serum  to  one  part  of  neutralized  veal 
broth,  to  which  one  per  cent  peptone,  one  per 
cent  beet  sugar,  and  one  per  cent  salt  is  added. 
These  Bacilli  Loeffler  regarded  as  important 
in  the  pathogenesis  of  diphtheria,  while  the  mi- 
crococci  he  considered  as  of  only  secondary 
importance. 

LITERATURE. 

BiRSCH-HiRSCHFELD  :  "  Archiv  fur  Heilkunde." 
1872,  p.  389. 

BUHL  :  "  Micrococci  of  Diphtheria.  Zeitschrift 
fur  Biologie,"  1867,  III. 

EBERTH  :  Zur  Kenntniss  d.  Bact.  Mykosen,  1872. 

FORSTER  :  "Miasmatische  Yerbreitung  d.  Diph- 
therie."  Wien.  med.  Wochenschr.  1881,  No.  24. 

GERHARDT  UND  KLEBS  :  "  Diphtheric,  ihre  para- 
sitare  Natur,Verhalteniss  des  localen  Prozesses  zur 
allgemeinen  Inflection,  Contagiositat,  Therapie 
(Chirurgie)  und  Prophylaxe."  Yerhandlung  des 
Congresses  fiir  innere  Medicin,  1882,  II.  Con- 
gress. 


206  THE  TECHNOLOGY  OF 

HOFFMANN:  "Ueber  Bakterien."  Botan.  Zei- 
tung,  1869. 

HUTER  u.  TOMMASI  :  (Micrococci  of  Diphtheria). 
Med.  Centralblt,,  1868,  p.  177,  531,  547. 

KLEBS  :  Archiv  f.  experiment.  Pathol.  u. 
Pharm.  IV. 

LETZERICH  :  "  Diphtherie-Pilze."  Yirchow's 
Archiv,  1872.  II :  "  Untersuchung  liber  die  mor- 
phologischen  Unterschiede  einiger  pathogenen 
Schizomyceten."  Archiv  f.  exper.  Path.  Bd.  XII. , 
H.  5,  p.  351  (1880). 

LIMMER  :  "  Uebertragung  der  Diphtherie  durch 
Hiihner."  Aerztl.  Int.  Bl.,  1881,  No.  31. 

LOEFFLER  :  "  Untersuchungen  liber  die  Bedeu- 
tung  der  Mikroorganismen  fiir  die  Entstehung  der 
Diphtheritis  beim  Menschen,  bei  Taube  und  beim 
Kalbe."  Mittheilung  a.  d.  kais.  Reichsgesund- 
heitsamt,  Bd.  2,  1884. 

NASSILOFF  :  "  Ueber  die  Diphtheritis."  Yir- 
chow's Archiv,  vol.  50,  p.  350. 

OERTEL  :  "  Experimentelle  Untersuchungen  liber 
Diphtherie."  Deutsche  Archiv  f.  klin.  Med. 
VIII.,  1871. 

TALAMON  (Cn.)  :  "Note  sur  le  microbe  de  la 
diphtherie."  Progres  med.  1881,  No.  7. 

WOOD  AND  FORMAD  :  "  The  nature  of  the  poison 
of  diphtheria."  Med.  Times  and  Gaz.,  1880,  Dec. 
4;  also  Bull.  National  Board  of  Health,  No.  17, 
1882. 


BACTERIA    INVESTIGATION.  207 

ERYSIPELAS. 

(Micrococci.) 

Fehleiserts  Method. —  Material  for  making  pure 
cultures  was  obtained  from  the  contents  of  a 
freshly  opened  erysipelas  bulla,  and  also  by  cut- 
ting out  a  bit  of  the  skin  of  the  affected  part, 
having  first  cleaned  it  with  ether  and  then  with  a 
corrosive  sublimate  solution.  These  he  placed  in 
nutritive  gelatine  and  stiffened  blood  serum,  and 
cultivated  according  to  general  rules  (q.  v.). 
With  the  pure  cultures  thus  obtained  he  vaccinated, 
among  others,  a  woman  (58  years  old)  who  was 
about  dying  with  multiple  sarcoma  of  the  skin,  and 
produced  a  typical  case  of  erysipelas. 

These  inoculations  upon  human  beings  were  jus- 
tifiable, because  they  were  undertaken  with  a  view 
to  cure  certain  tumors.  Thus  one  case  of  lupus, 
one  case  of  cancer,  one  case  of  sarcoma,  were  con- 
siderably affected  and  to  the  good  of  the  patient. 
Fehleisen  also,  in  several  instances,  succeeded  in 
second  inoculations,  after  the  lapse  of  a  few 
months.  The  vitality  of  this  microbe  is  destroyed 
by  three  per  cent  solutions  of  carbolic  acid,  or 
one  per  cent  solutions  of  corrosive  sublimate. 

LITERATURE. 

BAADER  :  "  Zur  Aetiologie  des  Erysipels," 
Schweis.  naturf.  Gesellsch.  Basel,  1875,  p.  314. 


208  THE  TECHNOLOGY   OF 

EHRLICH  :  "  Ueber  Erysipelas."  Langenbeck's 
Archiv,  Bd.  20,  p.  418.  " 

FEHLEISEN  :  I.,  "  Ueber  die  Ziichtung  der  Ery- 
sipelkokken  auf  kunstlichem  Nahrboden  und  ihre 
Uebertraffbarkeit  auf  den  Menschen."  Sitzungs- 

o  o 

ber.  d.  phys.  med.  Gesellsch  zu  Wurzburg, 
Jahrgang,  1883,  No.  1,  p.  9;  ibid.  No.  8,  Jahr- 
gang,  1881,  p.  126-128.  II.,  "  Die  Aetiologie  des 
Erysipelas."  Mit.  1  lith.  Tafl.,  Berlin  (Fischer), 
1883. 

LUKOMSKY  (W.)  :  "  Untersuchung  iiber  Erysi- 
pel."  Yirchovv's  Archiv,  vol.  60,  p.  418. 

ORTH  :  "  Untersuchnngen  iiber  Erysipel."  Arch, 
f.  experim.  Pathol.,  Bd.  L,  Hft.  III. 

RECKLINGHAUSEN  UND  SANKOWSKY  :  "  Ueber 
Erysipel."  Virchow's  Archiv,  Bd.  60,  p.  418. 

TILLMANS  :  "  Ueber  Erysipelas."  Verhand- 
lung  d.  dtsch.  Gesellsch.  f.  Chir.,  1878,  p. 
211. 

WOLFF  :  "  Ueber  Erysipelas."  Yirchow's  Ar- 
chiv, Bd.  81,  p.  173. 

FURONCLE. 
(Micrococci.) 

According  to  Pasteur's  description  these  microbes 
have  a  &amna-like  arrangement.  "  Couples  de 
deux  et  quatre  grains  et  paquet  de  ces  memes 
grains."  They  develop  within  less  than  six  hours 
after  being  put  into  the  culture  fluid.  He  con- 


BACTERIA    INVESTIGATION.  209 

siders  them  to  be  the  same  microbes  as  those  pro- 
ducing acute  infectious  osteomyelitis,  and  regards 
the  latter  disease  as  a  furoncle  of  the  marrow  of 
the  bone. 

Pasteur's  Method.  —  Pasteur  removed  from 
furoncles  small  amounts  of  pus,  which  he  cultivated 
in  chicken  broth  or  in  an  infusion  of  yeast  ("  1'eau 
de  levure  ")  at  35°  C.  He  inoculated  rabbits  with 
these  pure  cultures,  and  produced  boils.  This 
microbe  does  not  thrive  in  the  blood.  Stain  by 
general  methods. 

LITERATURE. 

GRAF  (FR.)  :  "  Die  Anticepsis  in  der  Ohrenheil- 
kunde."  Berlin,  klin.  Wochenschr.  1883,  No.  14, 
p.  209. 

LOWENBERG  (B.) :  I.,  " Untersuchungen  tiber 
Auftreten  und  Bedeutung  von  Coccobacterien  bei 
eitrigen  Ohrenflusse  und  iiber  die  durch  Gegen- 
wart  bedingten  therapeutischen  Indicationen." 
Zeitschr.  f.  Ohrenheilkunde,  X.,  1882.  II. ,  "Ke- 
cherches  sur  la  presence  de  micrococcus  dans 
Poreille  malade,  considerations  sur  le  r61e  des 
microbes  dans  la  furoncle  auriculaire  et  le  furoncu- 
lose  generale,  applications  therapeutique."  Compt. 
Eend.  T.  91,  p.  555,  1880. 

OGSTON:  "Ueber  Abscesse."  Arch.  f.  klin. 
Chir.,  Bd.  25. 

PASTEUR  :  "  Germes  in  Furoncles."  Compt. 
Kend.,  T.  90,  p.  1036. 


210  THE   TECHNOLOGY   OF 

V 

GONORRHOEA. 

(Micrococcus  gonorrhoea.) 

Neisser's  Method. —  Staining,  (a)  Spread  out 
the  secretion  in  the  thinnest  possible  layer,  upon  a 
very  thin  sterilized  cover-glass ;  this  is  done  best 
by  allowing  a  small  drop  to  flow  between  two  cover- 
glasses,  after  which  they  are  drawn  apart.  (6) 
Allow  the  preparation  to  dry  in  the  air,  heating  it 
slowly  up  to  120°  or  105°  C.,  at  which  temperature 
allow  it  to  remain  for  one  or  two  hours,  (c)  Prepara- 
tions must  be  well  stained.  All  the  basic  aniline 
dyes  may  be  used,  and  also  the  same  bases  united 
with  an  acid,  as  methyl-violet,  gentian-violet, 
fuchsin,  Bismarck-brown,  etc.,  but  none  are  so 
good  as  methyl-blue,  which  shows  a  great  affinity 
for  "  cocci."  Allow  the  prepared  cover-glasses  to  lie 
for  from  one-half  to  twenty-four  hours  in  a  con- 
centrated aqueous  solution  of  the  dye  stuff.  Wash 
in  absolute  alcohol,  pass  through  oil  of  cloves, 
and  mount  in  Canada  balsam.  Study  with  a  l-12th 
homogeneous  immersion  lens  and  an  Abbe's  con- 
denser. 

For  cultures  use  a  neutralized  peptone  beef- 
extract-gelatine,  a  blood-serum,  or  a  peptone- 
gelatine.  In  the  deep  parts  of  the  culture  medium, 
where  the  air  did  not  penetrate,  the  growth  was 
slow,  as  was  also  the  case  when  the  gelatine  was 
very  firm. 


BACTERIA    INVESTIGATION.  211 

Inoculation.  Dogs  have  immunity  from  this  dis- 
ease, inoculations  producing  a  simple  balanitis ; 
this  is  likewise  the  case  with  rabbits.  A  man 
forty-six  years  old,  into  whose  urethra  some  of  the 
pure  culture  was  injected  with  a  sterilized  syringe, 
had,  as  a  consequence,  a  true  gonorrhoea.  Inocu- 
lations were  made  upon  apes,  dogs,  cats,  and  rab- 
bits, on  the  conjunctiva,  cornea,  and  urethra,  by 
Neisser,  Leistikow,  Krause,  and  others. 

LITERATURE. 

AUFRECHT  :  "  Mikrokokken  in  den  inneren  Or- 
ganen  bei  Nabelrenen  EntzundungNeugeborener." 
Ctbl.  f.  d.  med.  Wiss.,  1883.  No.  16,  p,  273. 

BEAUVAIS  :  "  De  la  Balanite."  Gaz.  des  Hos- 
pitaux,  1874,  p.  867  e  876. 

BOCKHART  :  "  Beitrage  zur  Aetiol.  u.  Pathol. 
des  Harnrohren  Trippers."  Wiirzb.  phys.  med. 
Ges.,  Jahrgang,  1883.  No.  1,  p.  13.  No.  2,  p.  17. 

BOKAI  (A.)  :  "  Ueber  das  Contagium  deracuten 
Blennorrhoea."  Centralbl.  f.  d.  med.  Wiss.  1880. 
74. 

ESCHBAUM  :  "  Ein  Beitrag .  zur  Aetologie  der 
gonorrhoischen  Secrete."  Deutsch.  med.  Wochen- 
schr.  1883,  No.  13. 

FINKELSTEIN  :  Prager  med.  chir.  Presse,  May, 
1880. 

HAAB  :  "Der  Mikrokokkus  der  Blenorrh.  neana- 
torum."  Festschrift.  Weisbaden,  1881. 


212  THE   TECHNOLOGY  OF 

HIRSCHBERG  UND  KRAUSE  :  "  Untersucliungen 
iiber  Tripperbacterien  zur  pathologie  der  ansteck- 
enden  Augenkrankheiten."  Centralbl.  f.  pract. 
Augenheilkunde.  Sept.  1881.  Berl.  klin.  Wo- 
chenschr.  1882.  No.  32,  p.  500. 

KRAUSE  (F.).  "Die  Micrococcen  der  Blennor- 
rhoea  neanotorum."  Centralbl.  f.  pract.  Augen- 
heilkunde. Mai,  1882. 

LEISTIKOW.  "  Untersuchungen  iiber  Trip- 
perbacterien." Gesellsch.  d.  charit£  Aerzte  in 
Berlin,  16  Feb.  1882;  also,  Berl.  klin.  Wo- 
chenschr.  1882.  No.  32,  p.  500;  also,  No.  47,  p. 
719.  II.  "  Ueber  Bakterien  bei  den  venerisch- 
en  Krankheiten."  Charite  Ann.  VII.  (1882),  p. 
750. 

NEISSER  (A.):  "Die  Mikrokokken  der  Gonor- 
rhoe."  Dtsch.  med.  Wochenschr.  1882,  p.  279- 
282;  also  Med.  Centralbl.  1879,  No.  28. 

RETER  :  "Ueber  eine  der  Gonorrhoe  eigentiim- 
liche  Mikrokokkenform."  Centralbl.  f.  d.  med. 
Wissensch.  1879,  No.  28. 

EUCKER  (G.):  "Ueber  Polyarthritis  gonor- 
rhoica."  Dissert,  in  Berlin,  1880. 

WEIS  (F.):  "Le  microbe  du  pus  blennorrha- 
gique."  These  de  Nancy,  1880;  also  Annale  d. 
Dermatol.  1881.  Heft.  I. 

WELANDER  (E.)  :  "  Quelques  recherches  sur  les 
microbes  pathogenes  de  la  blenorrhagie."  Gaz. 
med.  de  Paris,  1884,  No.  23. 


BACTERIA    INVESTIGATION.  213 

OSTEOMYELITIS. 
Micrococci. 

Becker  was  the  first  to  make  pure  cultures  of 
this  microbe,  which  Pasteur  regards  as  identical 
with  that  found  in  furoncles. 

Krause's  Methods;  Cultures.  Remove  pus  with 
antiseptic  precautions  and  place  in  a  sterilized  test 
tube,  closing  the  same  with  cotton.  Use  for  cul- 
ture media  sterilized  coagulated  sheep's-blood 
serum  or  beef-juice-peptone-gelatine,  or  beef-juice 
agar  agar.  In  both  the  latter  mixtures  use  pep- 
tone siccum,  1-2  per  cent;  sodium  chloride,  0.5-1 
per  cent ;  sodium  phos.  sufficient  to  neutralize, 
gelatine,  5  per  cent;  or  agar  agar,  1-1.5  per 
cent.  Keep  object  glasses  bearing  cultures  in  a 
moist  chamber  and  in  the  breeding  oven.  The 
smell  of  the  cultures  of  this  microbe  is  character- 
istic ("  verdorbenen  Kleister")  like  spoiled  paste, 
and  is  apparent  the  moment  the  lid  of  the  moist 
chamber  is  raised.  Cultivate  also  on  slices  of 
boiled  potato,  and  notice  the  effect  on  milk  which 
is  rendered  sour.  Sterilize  the  milk  in  a  water- 
bath  at  100°  C.  and  inoculate  it  with  some  pure 
culture ;  it  becomes  lobbered  and  has  a  sour 
reaction. 

Staining.  Prepare  and  stain  according  to 
Gram's  general  method.  If  it  is  desired  to  give  a 
double  staining,  use  a  thin  solution  of  vesuvin. 


214  THE   TECHNOLOGY   OF 

Pasteur  considers  this  microbe  identical  with  that 
of  furoncles,  and  regards  osteomyelitis  as  a  furon- 
cle  of  the  marrow  of  the  bone. 

LITERATURE. 

BECKER  :  "  Osteomyelitiskokken."  Verein  f. 
innere  med.  zu  Berlin.  Dec.  17,  1883 ;  also, 
Deutsch.  med.  Wochenschr.  1883.  No.  46  and  No. 
52,  p.  816.  II.  :  Mikrokokken  der  Osteomy- 
elitis. Berl.  klin.  Wochenschr,  1883.  No.  52, 
p.  816. 

EBERTH  :  "Primare  infectiose  Periostitis."  Vir- 
chow's  Archiv,  Bd.  65,  p.  352. 

FEHLEISEN  :  (Panaritium)  Phys.  med.  Gesell- 
schaft.  Wurzburg.  1882,  p.  113-121. 

KOEHER  :  "Die  acute  Osteomyelitis."  Dtsch. 
Zeitschrift  f.  Chir.  Bd.  XL,  p.  96. 

KRAUSE  (F.)  :  "Ueber  einen  bei  der  acuten 
infectiosen  Osteomyelitis  des  Menschen  vorkom- 
menden  Mikrokokkus."  Fortschritte  der  Medicin, 
Bd.  2.  No.  8.  1884.  p.  261. 

LUCKE  :  "  Die  primare  infectiose  Knochenmark 
und  Knochenhautentziindung."  Dtsch.  Zeitschrift. 
f.  Chir.  Bd.  IV.,  p.  231. 

PASTEUR  :  Compt.  Rend.  T.  90,  p.  1036. 

RODET  (A.)  :  "  Etude  expe'rimentaic  sur  1'osteo- 
my^lite  infectieuse."  Compt.  Rend.  T.  99.  (Oct. 
6,  1884)  p.  569. 

ROSENBACH:  "Beitrage  zur  Kenntniss  der  Os- 


BACTERIA    INVESTIGATION.  215 

teomyelitis."      Dtsche.   Zeitschrift  f.   Chir.     Bd. 
X.,  p.  382. 

SCHULLER  :  "  Zur  Kenntniss  der  Mikrokokken 
bei  acuter  infectioser  Osteomyelitis,  Mikrokokken 
heerde  im  Gelenkknorpel."  Ctbl.  f.  Chir.  1881. 
No.  42. 

ACUTE    CROUPOUS    PNEUMONIA. 

Micrococci  pneumoniae  infect. 

Friedlander's  Method.  Cultures.  From  the 
lung  of  a  patient  who  has  died  of  this  disease  take 
small  pieces,  by  means  of  a  sterilized  wire,  and 
cultivate  in  blood-serurn  according  to  Koch's 
plan.  In  taking  the  material  it  must  first  be  seen 
to  that  the  lungs  are  perfectly  fresh,  i.  e.9  free 
from  all  appearances  of  decomposition ;  and  sec- 
ondly, that  contamination  from  without  is  avoided. 
Cut  pieces  from  the  pleural  surfaces,  or  with  a 
heated  knife  cut  out  a  piece  of  lung,  and  cut  suc- 
cessive slices  in  different  directions  from  this  until 
a  piece  is  obtained  which  is  completely  free  from 
external  contamination.  The  blood  serum  being 
inoculated  in  test  tubes  closed  with  sterilized 
cotton,  is  placed  in  a  thermostat  at  the  tempera- 
ture of  the  body.  Instead  of  blood  serum  Dr. 
Frobenius  used  an  infusion  of  meat,  peptone,  and 
salt.  Numerous  generations  should  be  made  to 
ensure  pure  cultures. 

The  pure  cultures  have  a  characteristic  "  nail " 


216  THE  TECHNOLOGY  OF 

shape  in  gelatine.  Very  good  cultures  in  grayish 
drops  may  be  made  on  slices  of  boiled  potato,  — 
these  give  the  characteristic  "  nail "  shape  when 
inoculated  back  on  to  gelatine. 

Inoculation.  Remove  a  bit  of  a  pure  culture 
by  means  of  a  sterilized  needle,  and  place  it  in 
a  watch-glass  previously  sterilized,  and  holding 
some  sterilized  distilled  water ;  by  mixing  this  a 
milky  fluid  is  obtained,  which  is  drawn  up  into 
a  previously  heated  Pravaz  syringe,  and  after 
cleaning  the  skin  of  the  animal  with  a  one  per  cent 
corrosive  sublimate  solution,  is  injected  into  the 
lung,  or  the  animal  is  made  to  breathe  the  spray  of 
the  same  fluid  produced  with  an  atomizer  properly 
sterilized,  and  operated  through  a  long  rubber 
tube,  in  order  that  the  investigator  may  not  inoc- 
ulate himself. 

Staining.  Pneumonia  Micrococci  are  charac- 
terized in  many  cases  by  a  peculiar  capsule. 
Water  and  alkalies  destroy  this,  and  Frankel  and 
Se'e  even  assert  that  the  capsule  does  not  belong  to 
pneumonia  Micrococci,  but  is  a  result  of  the  mode 
of  preparation  and  staining.  Gentian- violet  and 
fuchsin  stain  the  capsule  only  slightly,  while  giving 
the  Micrococci  a  deep  color.  Bismarck-brown  and 
methyl-blue  color  the  capsule  and  coccus  of  about 
alike  intensity.  Salvioli  and  Zaslein  obtained  the 
best  preparations  by  staining  with  a  mixture  of 
Bismarck-brown  and  methyl-violet. 


BACTERIA    INVESTIGATION.  217 

To  render  the  Micrococci  visible  in  tissues,  allow 
the  sections  cut  from  the  hardened  material  to 
remain  for  one  hour  in  aniline-gentian-violet  solu- 
tion [F.  19.]  at  45°  C.  Wash  in  iodo-iodide  of 
potash  solution  [F.  35  d],  pass  through  absolute 
alcohol,  oil  of  cloves,  and  mount  in  Canada 
balsam.  At  times  the  stained  Micrococci  give  up 
their  color  when  treated  with  alcohol  after  the 
potash  solution. 

To  render  the  capsules  visible  in  exudates  :  (1) 
prepare  cover-glasses  according  to  general  rules,  lay 
in  acetic  acid.,  color  in  gentian  violet  or  Bismarck 
brown ;  pass  through  alcohol,  oil  of  cloves,  and 
mount  in  Canada  balsam ;  or  (2),  allow  the  stain- 
ing fluid  to  act  for  twenty-four  hours;  or  (3), 
allow  the  cover-glasses  to  float  in  aniline  water- 
gentian-violet,  and  warm  to  steaming  for  one 
minute,  then  wash  one  half  minute  in  alcohol, 
and  study  in  distilled  water;  or  (4),  use  a  weak 
eosin  solution  for  twenty-four  hours;  or  (5), 
osmic  acid,  which  causes  the  contours  of  the  cap- 
sules to  stand  out  sharply. 

LITERATURE. 

BRUYLANTS  ET  VERRIERS  :  Bull,  de  1'Acad. 
Belg.  1880. 

EBERTH:    Dtsc'h.  Arch.  f.  klin.  Med.  Bd.  28. 

FRIEDLANDER  (C.)  :  I.  "  Die  Mikrokokken  der 
Pneumonie."  Fortschritte  der  Medicin,  Bd.  1, 


218  THE  TECHNOLOGY   OF 

1883.  No.  22,  p.  716.  II.  "Weitere  Bemerk- 
ungen  iiber  pneumonic  Micrococcen"  Ibid.,  Bd. 
2,  No.  10,  p.  340,  1884.  III.  "Ueber  die  Schi- 
zomyceten  bei  der  acuten  fibrinb'sen  Pneumonia." 
Yirchow's  Archiv,  Bd.  87,  p.  319.  IY.  "Den 
Micrococcus  der  crouposen  Pneumonie."  Berliner 
klin.  Wochenschr.  1883,  No.  48,  p.  752. 

FRIEDLANDER  u.  FROBENIUS  :  "  Der  Micrococcus 
der  crouposen  Pneumonie."  Berl.  klin.  Wochen- 
schr., 1883.  No.  48,  p.  752. 

GILES:  British  Med.  Journ.,  July  7,  1883. 

GRIFFINI  u.  CAMBRIA:  "Croupose  Pneumonie." 
Ctbl.  f.  d.  med.  Wiss.  1883,  p.  334. 

GUNTHER  u.  LEYDEN  (First  description  of)  : 
Sitzungsbericht  des  Yereins  fiir  innere  Med.,  Nov. 
20,  1882. 

JURGENSEN:  "Pneumonie  Micrococci"  (Con- 
gress fur  innere  Med.).  Fortschritte  der  Medicin, 
Bd.  2,  Heft.  9,  p.  333  (1884). 

KLEBS  :  Arch.  f.  experim.  Pathol.,  Bd.  IY. 

KLEIN  (E.)  :  "Ein  Beitrag  zur  Kenntniss  der 
Pneumokokkus."  Ctbl.  f.  d.  med.  Wiss.  1884, 
No.  30. 

KOCH  (Acute  phenomena  following  Recurrent 
fever)  :  Mittheilung.  a.  d.  kais.  Gesundheitsamt. , 
Berlin.  Bd.  1,  1881. 

LETZERICH  :  Morphologische  Unterschiede  eini- 
ger  pathogenen  Schizomyceten."  Archiv  f.  ex- 
perim. Pathol.,  Bd.  VII,  H.  5,  p.  351  (1880). 


BACTERIA    INVESTIGATION. 


MATROY  :  "  Pneumoniekokken."  Wiener  med. 
Presse.  1883.  No.  23,  24. 

MENDELSON  :  "  Pneumoniekokken."  Zeitschr. 
f.  klin.  Med.  1883,  VII.,  p.  206. 

POELS  (J.)  u.  NOLEN  (W.)  :  "Die  Mikrokok- 
ken  der  Pneumonie  des  Menschen  und  der  Lun- 
genseuche  der  Kinder."  Ctbl.  f.  d.  med.  Weiss. 
1884,  No.  9. 

PURJESZ  (S.)  :  "Die  Aetiologie  der  crouposen 
(fibrinosen)  Pneumonie."  Deutsch.  Arch.  f.  klin. 
Med.  XXXV.,  p.  301. 

SALVIOLI  u.  ZASLEIN  :  "  Ueber  den  Mikrokok- 
kus  und  die  Pathogenese  den  croupousen  Pneu- 
monie." Ctbl.  f.  d.  med.  Wiss.  1883,  No.  41, 
p.  721. 

SEE  (G.);  "  Sur  les  pneumonies  infectieuse  et 
parasitaires."  Comp.  Rend.,  Nov.  24,  1884.  T. 
99,  p.  931. 

STRASSMANN  (F.)  :  (Micrococci  of  Pneumonia)  . 
Stzb  Jenaisch.  Gesell.  f.  Naturwiss.  1883,  1884, 
p.  16,  17. 

VIERORDT  (O.)  :  "Zur  Kenntniss  des  Vorkom- 
mens  von  Spiralenbildung  irn  bronchial  Secret." 
Berl.  klin.  Wochenschr.  1883,  No.  29. 

ZIEHL  (F.)  :  "Ueber  das  Vorkommen  der  Pneu- 
moniekokken im  pneumatischen  Sputum."  Ctbl. 
f.  d.  med.  Wiss.  1883,  No.  25,  p.  433.  II. 
"Ueber  den  Nachweis  der  Pneumoniekokken  im 
Sputum."  Ibid.  1884,  No.  7. 


220  THE  TECHNOLOGY  OF 

RECURRENT  FEYER. 

Spirochcete  obermeyeri  (Colin). 

In  most  cases  the  microbes  of  this  disease  can- 
not be  studied  in  the  tissues,  since  the  /Spirilla 
seem  to  be  differently  constituted  from  other  Bac- 
teria, being  entirely  destroyed  by  acids,  alkalies, 
and  even  by  distilled  water.  They  act  much  more 
like  protoplasm  than  like  nuclei,  consequently  they 
are  not  stained  by  the  usual  methods  of  staining 
nuclei,  as  are  most  other  Bacteria.  Koch  is  the  only 
one  who  has  stained  them  (with  brown  aniline), 
and  photographed  them,  and  he  declares  the  iden- 
tification of  /Spirilla  in  hardened  organs  to  be  a 
very  different  matter. 

Rock's  Method  for  cover-glass  preparations  is  to 
dry  the  blood  upon  the  slide,  and  stain  with  fuch- 
sin,  methyl  violet,  or  gentian  violet,  or  methyl 
blue. 

Friedldnder's  Method  of  obtaining  the  microbes 
is  to  take  some  blood  from  the  patient  by  means 
of  the  cupping-glass,  and  allow  it  to  coagulate. 
The  Spirilla  gradually  gather  upon  the  surface  of 
the  clot,  often  in  large  groups  of  twenty  or  more 
twisted  up  in  a  glomerule.  They  will  retain  their 
vibratile  movements  for  hours,  even  for  days,  out- 
side of  the  body,  and  it  has  been  recently  shown 
(Albrecht)  that  they  also  increase  outside  the 
organism.  They  cause,  by  their  combined  move- 


BACTERIA    INVESTIGATION.  221 

ments,  strong  currents,  so  that  they  may  be 
examined  with  quite  low  powers.  They  are  only 
to  be  found  in  the  blood  during  febrile  periods. 

LITERATURE. 

ALBRECHT  :  I.  "  Zur  Entwickelung  der  Spi- 
rochaete  obermeyeri."  Archiv  klin.  Med.,  Bd. 
19,  1.  II.  "Eine  einfache  Method  zur  mikro- 
skopische  Untersuchung  des  Blutes  auf  Spirillen." 
Med.  chir.  Eundschau,  Bd.  XIX.  s.  508. 

BiRSCH-HiRSCHFELD :  "  Ueber  die  Recurrens- 
Spirochaeten."  Med.  Jahrb.  Bd.  166,  Heft.  2,  p. 
211. 

CARTER  :  (Production  of  Relapsing  fever  in 
monkeys,  by  the  inoculation  of  human  blood  con- 
taining Spirillum  Obermeyeri).  Lancet.  1879,  vol. 
1,  p.  84.  1880,  vol.  1,  p.  662. 

COHN  (F.)  :  "  Zur  weiteren  Kenntniss  des 
Febris  recurrens  und  der  Spirochaeten."  Deutsche 
med.  Wochenschr.,  1879,  No.  19. 

ENGEL  :  "  Ueber  d.  obermeyer'sch  Recurrens 
Spirillen."  Berl.  klin.  Wochenschr.,  1873,  p.  409. 

HEYDENREICH  :  "  Untersuchungen  iiber  d.  Para- 
siten  d.  Ruckfalltyphus."  Berlin:  1877;  also,  St. 
Petersburger  Wochenblatt,  1876. 

KOCH  (R.)  :  Deutsch.  med.  Wochenschr.  No. 
19,  1879. 

MANASSEIN  :  St.  Petersburger  Wochenblt.,  1876, 
No.  18. 


222  THE   TECHNOLOGY  OF 

MOLCHUTCHOWSKY  :  Ib id,  1876.     No.  6. 

OBERMEIER  :  "  Yorkommen  feinster,  eigene 
Bewegung  zeigender  Faden  im  Blut  von  Recur- 
rens-Kranken."  Med.  Centralbl.  Bd.  XI,  10, 
1873.  Berl.  med.  Gaz.  Marz,  1873 ;  Berl.  klin. 
Wochenschr.,  1873,  p.  152  and  391. 

WEIGERT  :  "  Bemerkungen  iiber  die  obermeier'- 
schen  Recurrens-faden."  Dtsch.  med.  Wochenschr., 
1876,  p.  471-498. 

YELLOW   FEVER. 
Micrococci  (Cryptococcus). 

Friere's  ethod  was  to  cultivate  the  microbes 
obtained  from  the  blood  of  yellow-fever  patients 
according  to  the  general  methods,  and  to  attenuate 
the  cultures  by  heat  in  a  manner  similar  to  that 
employed  by  Pasteur  for  anthrax.  In  November, 

1883,  he  received  permission  from  the  Emperor  of 
Brazil  to  vaccinate  human  beings ;  this  he  did  to 
the  extent  of  several  hundreds,  giving  them  the 
disease  in  a  benign  form.     It  is  not  known  how 
long  the  immunity  conferred  by  inoculation  lasts, 
but  at  first  it  is  absolute. 

LITERATURE. 

FRIERE  (D.)  ET  REBOUGEON  :  "  Le  microbe  de 
la  fievre  jaune.  Inoculation,  Preventive."  Compto 
Eend.  de  1'Acad.  de  Sc.  de  Paris,  T.  99,  Nov.  10, 

1884,  p.  804. 


BACTERIA    INVESTIGATION.  223 

LECAILLE  :  "  Le   microbe   de  la  fi£vre  jaune." 
Journal  de  Micrographie.     T.  8,  p.  75. 


MISCELLANEOUS  PATHOGENIC  BACTERIA. 
Not  finding  any  special  methods  given  for  the 
study  of  the  following  forms,  which  can  be  studied 
by  the  general  rules  given  in  Chapter  I,  or  by  any 
of  the  numerous  modifications  of  these  rules  given 
for  the  various  "specific"  microbes,  I  will  simply 
enumerate  them,  and  give  their  literature,  that 
further  research  in  regard  to  them  may  be  incited 
and  facilitated. 

CONTAGIOUS  SEPTICAEMIA. 

Micrococcus  septicus  (Comsr). 

Besides  the  septicaemia  produced  by  M.  septicus 
(Microsporon  septicum  Klebs),  Sternberg  claims 
to  have  produced  the  disease  in  rabbits  by  the  Mi- 
crococci  obtained  from  his  own  saliva,  while  Koch 
produced  a  septicaemia  in  mice  by  the  injection  of 
a  putrescent  meat  broth  under  the  skin  of  the 
back.  Extensive  gangrene,  with  much  cedema- 
tous  exudation,  follo~wed,  and  death  ensued  in 
two  days  and  a  half.  The  blood,  the  capillaries 
of  the  kidney,  and  the  enlarged  spleen,  contained 
numerous  oval  Micrococci,  singly  as  dumb-bells, 
and  in  zoogloea. 


224  THE   TECHNOLOGY   OF 


LITERATURE. 

ARDEIS^E  :  "Les  Microbes,  les  Miasmes,  et  les 
Septicaemies."  Paris,  1882. 

BILLROTH  :  "  Untersuchungen  iiber  die  Vegeta- 
tions formen  der  Coccobacteria  Septica."  Berlin, 
1874,  pp.  200. 

BINZ  :  "  Ueber  den  Einfluss  innere  Arzuiittle  auf 
die  Septicaemieund  andere  Infectionskrankheiten." 
Wien,  Med.  Pr.,  1881,  No.  38. 

DEGAGNY  (C.) :  "  Sur  le  micro-organisme  d'une 
septice'mie  observee  chez  1'homme  et  le  mouton." 
Soc.  de  Biol.  17  Mai,  1884. — Journal  de  Micro- 
graphie,  1884,  p.  348. 

GAFFKY  :  "  Experiment  Erz.  Septicaemia."  Mitt- 
heilung.  a.  d.  kais.  Gesundheitsamt,  Bd.  1.,  1881, 
Berlin. 

KOCH  (R.)  :  "Untersuchungen  iiber  die  Aeti- 
ologie  d.  Wundinfections-Krankheiten."  Leipzig, 
1878. 

LITTEN  :  "  Septiceemie."  Danziger  Naturf.  Yers., 
1881. 

EOSENBERGER:  I.  "Ueber  Septicaamie."  Phys. 
med.  Gesellsch.  zu  Wiirzburg,  1882,  p.  41,  45. 
II.  "  Experimentelle  Studien  iiber  Septicaemie." 
Ctbl.  f.  d.  med.  Wiss,  1883,  No.  4. 

STERNBERG,  (G.  M.)  :  "  Fatal  Form  of  Septi- 
caemia in  the  Rabbit,  produced  by  the  Sub- 
cutaneous injection  of  Human  Saliva."  Report, 


BACTERIA    INVESTIGATION.  225 

Nat.  Board  of  Health  Bull.  April  30,  1881,  22  pi., 
Ipl. 

TIEGEL  :  "  Ueber  die  fiebererregende  Eigen- 
schaft  des  Microsporon  septicum."  Dissertation, 
Bern.  1871.  See  also,  Correspondenzblatt  fur 
Schweizer  Aerzte  1871,  p.  275. 

WAGNER  (P.)  :  "Ueber  Aetiologie  und  Symp- 
tomatologie  der  kryptogenetischen  Septicopyae- 
mia."  D.  Archiv f.  klin.  Med.Bd.  XXVIII,  p.  562. 

RHEUMATIC  ARTHRITIS. 

Micrococcus  rheumarthritis. 
LITERATURE. 

LEYDEN  (M.  H.) :  "  Gelenkenrheumatismus  Mi- 
cro-organismen  (Streptococcus),"  Dtsch.  med. 
Wochenschr.,  1882,  p.  656. 

KUSSMAUL  :  "Mikrokokken  bei  Gelenkrheuma- 
tismus,"  Roser's  und  Wunderlich's  Archiv  f.  phys- 
iol.  Heilkunde,  Bd.  XI.,  p.  626. 

ENDOCARDITIS  ULCEROSA. 
LITERATURE. 

EBERTH  (C.  J.) :  "  Ueber  diptheritische  Endo- 
carditis." Virchow's  Archiv,  vol.  57,  p.  228. 

HEIBERG  :  Ein  Fall  von  Endocarditis  ulcerosa 
puerperalis,  mit  Pilzbildung  im  Herzem  (Mycosis 
endocardii).  Yirchow's  Archiv,  vol.  56,  p.  407. 

KLEBS  :  "  Weite^e  Beitrag  zur  Entstehungsge- 
schichte  der  Endocarditis,"  Archiv  f.  experirn. 
Pathol.,  Bd.  IX.,  1873,  p.  52. 


226  THE   TECHNOLOGY   OF 

KOESTER  :  "  Die  embolische  Endocarditis." 
Yirchow's  Archiv,  vol.  72,  p.  257. 

MAIER  (R.) :  "  Ein  Fall  von  primarer  Endo- 
carditis diptheritica."  Virchow's  Archiv,  vol.  62, 
p.  145. 

INFECTIOUS  WOUND  DISEASES. 

Micrococci. 
LITERATURE. 

KOCH  (R.)  :  "Ueber  d.  Aetiologie  d.  Wundinfec- 
tions-krankheiten,"  Leipzig,  1878,  Ctbl.  f.  med., 
Wiss.,  1879,  p.  175. 

ROSEKBACH  (F.  J.) :  I.  "  Micro-organismen  bei 
den  Wundinfectionskrankheiten  der  Menschen," 
Wiesbaden  (J.  F.  Bergmann),  1884.  II.  "Unter- 
suchungen  iiber  die  kleinster,  lebender  Wesen  zu 
den  Wundinfectionskrankheiten  der  Menschen," 
Wiesbaden,  1885  ;  III.  "  Ueber  einige  fundamentale 
Fragen  in  der  Lehre  von  der  chirurgischen  Wund- 
infectionskrankheiten. Giebt  es  verschiedene  Arten 
der  Faulniss  ?  "  Dtsch.  Zeitschr.  f.  Chir. ,  Bd.  XVI. , 
p.  342-368. 

VERNEUIL  (A.)  :  "De  Fauto-inoculation  trau- 
matique,"  Revue  de  Chir.,  Bd.  III.,  No.  12,  Dec. 
12,  1883,  p.  921-952. 

WASSILIEF  (N.  P.) :  "  Beitrage  zur  Frage 
iiber  die  Bedingungen  unter  denen  es  zur  Ent- 
wickelnng  von  Mikrokokken  Colonien  in  den 
Blutgefassen  kommt."  (Aus  deni  Strasburger 


BACTERIA    INVESTIGATION.  227 

pathol.  Inst.)      Ctbl.    f.    d.    med.   Wiss.,    1881, 
No.  52. 

WOLFF  :  "  Zur  Bacterien  Lehre  bei  accidentellen 
Wundkrankheiten,"  Yirchow's  Archiv  f.  pathol. 
Anat.  u.  PhysioL,  Bd.  81,  p.  139. 

VARIOLA. 

Micrococcus  variolce  et  vaccince  (Colin). 

To  positively  connect  the  Micrococci  in  this  case 
with  the  disease  is  a  matter  still  unaccomplished. 
A  series  of  accurate  and  careful  culture  experi- 
ments should  be  made,  absolutely  pure  cultures 
obtained,  and  re-inoculations  made  to  ascertain 
whether  the  disease  can  be  reproduced.  To  stimu- 
late such  a  research,  the  Grocers'  Company  of 
London  have  offered  a  prize  of  £1000. l 

1  The  Worshipful  Company  of  Grocers'  First  Quadrennial  Dis- 
covery-Prize of  £1000,  1883-1886,  in  aid  of  original  research  in 
sanitary  science,  have  announced  the  following  problem :  — 

"  To  discover  a  method  by  which  the  vaccine  contagion  may  be 
cultivated  apart  from  the  animal  body,  in  some  medium  or  media 
not  otherwise  zymotic  —  the  method  to  be  such  that  the  contagion 
may,  by  means  of  it,  be  multiplied  to  an  indefinite  extent  in  suc- 
cessive generations,  and  that  the  product  after  any  number  of  such 
generations  shall  (so  far  as  can  within  the  time  be  tested)  prove 
itself  of  identical  potency  with  standard  vaccine  lymph." 

The  prize  is  open  to  universal  competition,  British  and  foreign. 
Competitors  for  the  prize  must  submit  their  respective  treatises 
on  or  before  the  31st  of  December,  1886;  and  the  award  will  be 
made  as  soon  afterward  as  the  circumstances  of  the  competition 
shall  permit  —  not  later  than  the  month  of  May,  1887.  In  relation 
to  the  Discovery-Prize,  as  in  relation  to  other  parts  of  the  com- 
pany's scheme  in  aid  of  sanitary  science,  the  court  acts  with  the 


228  THE  TECHNOLOGY   OF 


LITER  ATTJKE. 

CARPENTER:  (Vaccine),  vid.  "  Paper  on  the 
Germ  Theory  of  Disease  from  a  Natural  History 
Point  of  View."  British  Assoc.,  1883. 

CHATJVEAU  :  "  Nature  du  virus-vaccin,  D£- 
termination  expe'rimentale  des  elements  qui  con- 
stituent le  principe  actif  de  la  serosite  vaccinale 
virulente."  Compt.  Rend.  T.  66  (1868),  p.  289, 
317,  359,  948.  Chauveau  was  the  first  to  prove 
experimentally  that  the  active  principle  of  the 
virus  of  vaccinia  and  variola  is  a  particulate  non- 
diffusible  substance. 

COHN  :  "  Micrococcus  vaccinae,"  Virchow's  Ar- 
chiv,  1872. 

CORNIL  ET  BABES  :  "  Note  sur  le  siege  des  bac- 
t^ries  dans  le  variole,  la  vaccine  et  Terysipele,"  8°, 
6  pp.,  Paris  ( Alean-Levy) ,  1884,  Extr.  de  1'Union 
med.  VIII.,  1883. 

JOLYET  :  "  Sur  1'etiologie  et  la  pathologe*nie  de 
la  variole  du  pigeon,  et  sur  le  developpement  des 
microbes  infectieux  dans  lymph."  Compt.  Rend. 
T.  92,  p.  1522. 

KLEBS  (E.)  :  I.  "  Der  Micrococcus  der  Variola 

advice  of  a  scientific  committee,  which  at  present  consists  of  the 
following  members:  — John  Simon,  C.B.,  F.R.S.;  John  Tyndall, 
F.R.S.;  John  Burdon  Sanderson,  M.D.,  F.R.S.;  and  George 
Buchanan,  M.D.,  F.R.S. 

All  communications  on  the  subject  are  to  be  addressed  to  the 
clerk  of  the  Grocers'  Company,  Grocers'  Hall,  London,  E.G. 


BACTERIA  INVESTIGATION.  229 

und  Vaccine,"  Archiv  f.  experim.  Pathol.,  Bd.  X., 
Hft.  3  and  4,  also  Bd.  13,  p.  284. 

MARCHAND  :  "  Les  virus-vaccins."  Eevue  My- 
cologique,  IV.  Annee,  Avril,  1882. 

POHL-PINCUS:  "Vaccination."     Berlin,  1882. 

SANDERSON  (BUKDON)  :  Report  on  the  Intimate 
Pathology  of  Contagion. 

STRAUSS  :  "  Vaccinal  Micrococci."  Socie^  de 
Biol.  de  Paris.  1882. 

STROPP  :  "  Vaccination  u.  Mikrokokken."  1874. 

TSCHAMER  :  "  Ueber  das  Wesen  des  Contagiums 
der  Variola,  Vaccine,  und  Varicella."  Aerztl. 
Verein,  Steiermark,  1880,  Sept.  8. 

WEIGERT  :  "  Ueber  Bakterien  in  der  Pocken- 
haut."  Ctbl.  f.  d.  med.  Wiss.,  1871,  No.  49. 

INFLUENZA  EPIDEMICA. 

Micrococcus  influenzce. 
LITERATURE. 

LETZERICH  :  Morpho.  Unterschiede  einigen  path. 
Schizomyceten,  Archiv  f.  experiin.  Pathol.,  Bd. 
XII,  Hft.  5,  p.  351  (1880). 

MENINGITIS. 

Micrococci. 
LITERATURE. 

AUFRECHT  :  "  Zwei  Falle  von  Meningitis  cere- 
brospinalis"  (Mikrokokken).  Deutsche,  med. 
Wochenschr.,  1880,  No.  4. 


230  THE  TECHNOLOGY   OF 

GAUTIER  :  M  Micrococcen  bei  Meningitis,"  Gaz. 
med.  de  Paris,  1881,  Vol.  XXXVII. ,  7. 

LEYDEN  :  "  Die  Mikrokokken  dor  cerebrospinal- 
meningitis."  Ctbl.  f.  klin.  Med.,  1883,  No.  10. 

MEASLES. 

Micrococci. 

LITERATURE. 

KEATING  ( J.  M.) :  "  The  presence  of  micrococcus 
in  the  blood  of  malignant  Measles."  Phila.  Med. 
Times,  No.  384. 

KLEBS  :  I.  "Der  Micrococcus  der  Masern," 
Sitzungsber.  d.  phys.  med.  Gesellch.  zu  Wiirzburg, 
fiir  1873,  p.  VII.  II.  " Micrococcen  als  Krankheit- 
ursache,"  Berl.  klin.  Wochenschr.,  1873,  p.  116. 

PHLEGMON. 

Diplococci. 
LITERATURE. 

CORNIL  ;  "  Sur  1'anatomie  pathologique  du  phleg- 
mon et,  en  particulier,  sur  le  siege  des  bacteries 
dans  cette  affection."  Compt.  Rend,  de  1'Acad.  de 
sc.  de  Paris,  1883,  T.  97,  p.  1594. 

SCARLET  FEVER. 
LITERATURE. 

GAZE  u.  FELTZ  :  "Malad.  Infect."     1872. 
HAHN  (E.)  :"  Demonstration  des  Scharlachs  auf- 
tretenden  Schizomyceten,"  Berl.  med.   Gesellsch, 


BACTERIA    INVESTIGATION.  231 

Marz,  1883  ;  Berl.  klin.  Wochenschr.,  No  38, 
1882,  p.  583. 

KLEIN  (E.)  :  "  Eeport  of  the  Medical  Officer 
of  the  Privy  Council  for  1876." 

McKENDRiCK  :  Brit.  Med.  Journ.,  1872. 

PoHL-PiNCUS :  "Mikrokokken  in  dem  Epider- 
misschuppen  von  Scharlochkranken."  Ctbl.  f.  d. 
med.  Wiss.,  1883,  No.  36. 

MOLLUSCUM  CONTAGIOSUM. 
LITERATURE. 

ANGEL AUS  :  "  Zur  aetiologie  von  Molluscum 
contagiosum,"  Abth.  f.  med.  Wiss.,  1881,  3. 

DOMENICO  (M.)  :  "  Sul  Bacillo  del  mollusco  con- 
tagioso."  Atti.  Acad.  Lincei,  Transunti.  Y.,1880, 
p.  77.  —  Gazetta  Medica  di  Roma.,  19,  20,  1880. 

DILATATION     AND     OTHER     DISEASES      OF     THE 
STOMACH. 

Sarcina  ventriculi  (Goodsir). 
LITERATURE. 

EBERTH  :  w  Ueber  Sarcina  ventriculi."  Virchow's 
Archiv,  1858,  Bd.  XIII.,  p.  522. 

KLEBS  :  "  Ueber  infectiose  Magenaffectionen." 
Allgem.  Wiener  med.  Zeitung,  1881,  No.  29.35. 

LOSDORFER  :  "  Ueber  Sarcina  ventriculi."  Med. 
Jahrb.,  1871,  H.  3. 

SURINUAR  :  "De  Sarcina  ventriculi,"  Arch.  Neer- 
land,  1866;  Botan.  Zeitung,  1866,  p.  269. 


232  THE  TECHNOLOGY   OF 

YIRCHOW  AND  COHNHEIM  i  "  Ueber  Sarcina  ven- 
triculi,"  Virchow's  Archiv,  Bd.  10  and  33. 

AMAEMIA  PERNICIOSA. 

(Micrococci). 

FRANKENHAUSER  :  Cthl.  f.  d.  med.  Wiss.  No.  4, 

1883. 

ULCERATIVE  STOMATITIS  IN  THE  CALF. 

(Bacilli.) 

LINGARD  (A.)  AND  BATT  (E.)  :    Lancet,  May, 
1883. 

CATTLE  PLAGUE. 

(Bacilli.) 

ARCHANGELSKI  :  Ctbl.    f.    d.    med.    Wiss.    No. 
18,  1883. 

LANZI  :  "Microbe  di  Morbillo."    Bull.    Accad. 
Med.  Roma.  IX.,  1883,  No.  7. 

METZDORF  :   (Bacillus  of  Cattle  Plague).    Bied. 
Centr.,  1884,  p.  419,   420.     Journ.    Chem.    Soc. 
(Abstr.),  xlvi.  (1884),  p.  1398. 
ROLL  :  «  Die  ThierseuchenV     Wien,  1881. 
ROLOFF  (T.)  :    Archiv  f.  wiss.  u.  prakt.  Thier- 
heilkunde.,  IX.,  1883. 

ACUTE  YELLOW  ATROPHY  OF  THE  LIYER. 

(Micrococci.) 

EPPINGER  :  Prager  Viertelj.     1875. 
BALZER  (F.)  :     "  Parasitisme  du  Xanthelasmo 


BACTERIA    INVESTIGATION.  233 

et  de  Picture   grave."     Archiv  d.  phys.  nor.  et. 
path.  1882,  X.,  p.  307. 

DYSENTERY. 
(Micrococci  and  Bacilli.) 

PRIOR  :  "Die  Mikrokokken  bei  der  Dysenterie." 
Ctbl.  f.  klin.  Med.  1883,  No.  17. 

LINGARD  (A.)  :  Klein's  "Micro-organisms  and 
Disease."  1885,  p.  128. 

OZ^ENA. 

(Diplococcus.) 

LOWENBERG  :  "  Die  Natur  und  die  Behandlung 
der  Ozsena."  Union  Med.  No.  22-25,  1884. 
Deutsch.  med.  Wochenschr.  1885,  1,  2. 

FOOT  ROT. 

TOUSSAINT  :  "  Sur  la  culture  du  microbe  de  la 
clavele'e."  Compt.  Rend.  XC1L,  1881,  p.  362-4. 

VERRUGA  PERUANA. 

(Bacilli.) 

IZQUIERDO  (Y.)  :  "  Spaltpilze  bei  der  Verruga 
peruana."  Yirchow's  Archiv,  Bd.  99,  Hft.  3, 
p.  411. 

DIABETES. 

FRIEDRICH  (N.)  :  "  Ueber  das  constante  Yor- 
kommen  von  Pilzen  bei  Diabetischen."  Yirchow's 
Arch.,  vol.  XXX.,  p.  476,  1864. 


234  THE  TECHNOLOGY   OF 

CHYLUKIA. 

(Bacilli. ) 

WILSON  (A.)  :  "A  case  of  chyluria  caused  by 
Bacilli,  with  cultivation  experiments."  Brit.  Med. 
Journ.,  1884,  Dec.  6. 

FCETID  FEET. 

THIN  (G.)  :  "  Bacterium  foetidum:  an  organism 
associated  with  profuse  sweating  of  the  soles  of 
the  feet,  cultivated  in  vitreous  humor."  Proc. 
Eoy.  Soc.,  XXX.  (1880),  p.  473. 

MALLEUS  HUMIDUS. 

DIRNER  :  "Em  an  einem  Menschen  beobachten 
Fall  von  Malleus  humidus." 

KOZAHEGGI  :  "  Bakterien  im  Eiterpusteln  bei 
Malleus  humidus."  Pestes  med.  chir.  Presse. 
1883,  No.  35. 

PYAEMIA. 

BELZOW  (A.)  :  "Zur  Frage  der  Mikroorganis- 
men  bei  Pysemia."  Ctbl.  f.  d.  med.  Wiss.  1884, 
No.  22,  p.  370. 

KOCH  (R.)  :  "Untersuchungeniiber  die  Aetiolo- 
gie  d.  Wundinfections-Krankheiten.  Leipzig, 
1878. 

DISEASES  OF  INSECTS. 
LITERATURE. 

BECHAMP  :  "  Microzyma  bombycis.  Compt. 
Rend.  T.  64,  1867. 


BACTERIA    INVESTIGATION.  235 

BURRILL  (T.  J.)  :  Disease  of  insect  Blissus 
leucopterus.  Amer.  Naturalist,  XVII.,  1883,  p. 
319,320.  Named  the  microbe  "  Micrococcus  in- 
sectorum." 

FORBES  (S.  A.)  :  "Bacterial  Parasite  of  the 
chinch-bug  (Micropus  leucopterus)."  Amer.  Natu- 
ralist, XVI.,  1882,  pp.  824.  II.  "Experiments 
with  diseased  caterpillars  (Schlaffsucht),  claims 
the  microbe  found  in  Datana  ministra  is  identical 
with  Micrococcus  bombycis.  He  also  found  Mi- 
crococci  in  Datana  angusii  and  in  the  cabbage- 
worm,  Pier  is  rapce. 

PASTEUR  (L.)  :  "  Etude  sur  la  maladie  des  vers 
a  soie,  moyen  pratique  assure*  de  la  combattre  et 
d'en  prevenir  la  retour."  1870,  L,  p.  228. 

BACTEPJA  IN  PLANT  TISSUES. 

Ducleaux  studied  the  effect  of  microbes  upon 
germination  by  planting  peas  and  beans  in  soil 
previously  sterilized  and  moistened  with  milk  also 
sterilized.  There  was  no  germination,  and  at  the 
end  of  two  months  the  milk  showed  no  indication 
of  alteration,  the  whole  apparatus  in  which  the 
experiment  was  conducted  being,  of  course,  pro- 
tected against  contamination  from  the  air. 

Ralph's  Method  for  demonstrating  the  presence 
of  Bacilli  in  the  cells  of  water-plants  (  Vallisneria, 
etc.).  A  thin  section  of  the  cuticle  of  the  leaf 
should  be  sliced  off  and  placed  on  a  slide,  with 


286  THE  TECHNOLOGY  OF 

the  cuticular  surface  next  the  cover ;  the  slide 
should  then  be  placed  on  a  rest,  with  the  cover 
downwards  or  towards  the  table,  and  allowed  to 
remain  there  for  five  minutes  at  least,  to  allow  the 
organisms  to  fall  on  the  cuticular  walls  of  the 
cells,  then  examine  under  a  quarter-inch  objective. 
The  Bacteria  must  be  looked  for  in  the  quadrate 
cells,  where  they  may  be  seen  moving  about  the 
chlorophyll-grains  even  when  cyclosis  is  going  on. 
After  the  lapse  of  some  minutes  they  will  gravi- 
tate out  of  sight,  or  be  found  heaped  together  at 
the  lower  end  (or  apparent  upper  end)  of  the  cell. 
It  is  this  circumstance  which  has  prevented  any 
recognition  of  their  presence  heretofore  ;  they  are 
rarely  if  ever  seen  in  the  long,  deep-seated  cells 
which  exhibit  cyclosis  so  well  in  Vallisneria. 

LITERATURE. 

BATILIN  :  "Bacterien  inficirte  Weizen  und  Mais- 
korner."  Botan.  Zeitung,  1882,  p.  28. 

BURRILL  (T.  J.)  :  "  Micrococcus  toxicatus,  the 
peculiar  poison  of  Rhus."  Amer.  Naturalist, 
XVIL,  1883,  p.  319.  II.  "Disease  of  Koots  of 
Strawberry  Plants."  Bacillus  shown  at  regular 
meeting  of  State  Microscopical  Society  of  Illinois, 
Feb.  8,  1884. 

DUCLAUX:  "Effect  of  presence  of  microbes 
upon  germination."  Meeting  of  Acad.  of  Sci., 
Paris,  Jan.  5,  1885. 


BACTERIA    INVESTIGATION.  237 

RALPH  (T.  S.)  :  "Living  Bacilli  in  the  cells  of 
Vallisneria  and  Anacharis."  Journal  of  Mi- 
croscopy, iii.  (1884),  p.  17. —  Proc.  Roy.  Soc. 
Victoria,  10  May,  1883. 

REINKE  u.  BERTHOLD  :  (Plant  pathology).  Un- 
tersuchungen  aus  dem  botan.  Laboratorium  in 
Gottingen.  Hft.  I. 

WALKER  :  "  Yellow  disease  of  Hyacinths."  Bo- 
tan.  Centralblatt.  Bd.  14,  p.  315. 


BACTERIA  INVESTIGATION. 


PART  III. 
FORMULARY. 

1.  BERGMANN'S  OR  BUCHOLTZ'S  FLUID. 

Distilled  water 100      ccm. 

White  rock  candy 10        " 

Tartrate  of  ammonia 1        ** 

Phosphate  of  lime 0.5     " 

2.  BISMARCK-BROWN. 

This  takes  the  lead  of  all  aniline  dyes,  in  that  it 
is  not  so  easily  washed  out.  The  preparations  may 
also  be  mounted  in  glycerine,  which  is  not  the 
case  with  other  aniline  dyes.  It  is  also  especially 
adapted  to  photography.  Weigert  makes  a  satu- 
rated solution  with  boiling  distilled  water,  or  one 
in  which  there  is  very  little  alcohol ;  after  cooling, 
filter,  set  aside,  and  allow  to  settle.  After  stain- 
ing, wash  the  sections  for  a  short  time  in  absolute 
alcohol.  Whole  pieces  may  be  stained  at  once 
before  cutting  by  using  an  alcoholic  solution  of 
Bismarck-brown.  Baumgarten  used  a  concentrated 
(one  per  cent.)  acetic  acid  solution. 


BACTERIA   INVESTIGATION.  239 

3.  GLYCERINE  ANILINE-BROWN. 

Used  by  Koch  for  photographing.  Make  a 
concentrated  solution  of  aniline-brown  in  equal 
parts  of  glycerine  and  water.  Filter  from  time 
to  time. 

4.  ANILINE  OIL. 

This  is  a  mixture  of  two  similar  aniline  bases  — 
toluidin  and  pseudotoluidin.  It  is  a  yellowish, 
oily  fluid,  whose  saturated,  watery  solution  allows 
of  the  dissolving  of  more  coloring  matter  than  the 
dilute  solution  of  caustic  potash  first  used  by 
Koch.  When  it  is  used  in  connection  with  the 
aniline  dyes,  the  latter  may  be  added  directly  to 
it,  and  the  two  shaken  together,  the  mixture  being 
allowed  to  stand  and  steep  in  an  oven  at  a  temper- 
ature of  about  95°  F.  for  from  fifteen  minutes  to 
twenty-four  hours  before  using,  or  a  concentrated 
alcoholic  solution  of  the  coloring  material  is 
poured  into  a  ten  per  cent  aniline-oil-water.  The 
colors  best  adapted  for  use  in  connection  with 
aniline  oil  are  fuchsin,  methyl-violet  and  gen- 
tian-violet. These  solutions,  especially  at  a 
temperature  of  95°  F.,  will,  in  from  two  to 
twenty-four  hours,  stain  those  Bacteria  which, 
as  a  rule,  show  such  stubborn  resistance  to 
staining  fluids. 


240  THE  TECHNOLOGY  OF 

5.  ANILINE  WATEE. 

To  make  an  aniline  water  that  is  permanent 
and  does  not  need  to  be  filtered,  take  — 

Aniline  oil  or  Toluidin 3  ccm. 

Alcohol 7    " 

Dissolve  and  add  water 90    " 

(Fraenkel.) 

6.  ANILINE  YELLOW. 

Aniline  yellow 0.2  ccm. 

Distilled  water 10       " 

7.  ACIDULATED  FLUIDS  FOR  DECOLORIZING. 

Ehrlich  and  Balmer-Fraenzel  used  a  (33  per 
cent)  watery  solution  of  nitric  acid. 

Friedlander  used  nitric  acid  and  alcohol  (33J 
per  cent). 

Glacial  acetic  acid  is  very  well  adapted  for 
clearing  up  sections  in  which  the  Bacteria  are  to 
be  studied  without  staining,  rendering  them  very 
distinct. 

Hydrochloric  acid,  one  part,  to  70  per  cent 
alcohol,  one  hundred  parts.  Hydrochloric  acid, 
20  ccm. ;  90  per  cent  alcohol,  100  ccm. ;  water, 
20  ccm. 

Coze  and  Simon  recommend  the  following  mix- 
ture (Brurfs). 

Nitric  acid 5  grammes. 

Acetic  acid 10 

Distilled  water  .    .    55 


BACTEEIA    INVESTIGATION.  241 

8.  ETHER. 

In  tissues  containing  much  fat,  this  may  be  re- 
moved by  the  use  of  ether  in  connection  with 
absolute  alcohol.  Allow  the  preparation  to  lie  in 
the  alcohol  until  it  is  entirely  dehydrated,  then 
place  in  the  ether.  If  it  causes  the  ether  to 
become  turbid,  it  lias  not  been  sufficiently  long  in 
the  alcohol.  After  they  have  remained  for  some 
time  in  the  ether  (or  chloroform)  place  again  in 
alcohol,  and  then  in  water. 

9.  ALCOHOL. 

All  alcohol  used  in  the  treatment  of  Bacteria, 
especially  in  connection  with  staining,  must  be 
perfectly  pure  and  without  any  acid  reaction. 

10.  CANADA  BALSAM. 

It  is  important,  for  mounting  stained  Bacteria,  to 
use  Canada  balsam  free  from  chloroform,  as  Orth 
and  others  testify  that  it  removes  the  color  from 
the  microbes. 

Baumgarten  recommends  equal  parts  of  Canada 
balsam  (free  from  chloroform)  and  oil  of  cloves. 

11.   CIIRYSOIDIN. 

Make  a  concentrated  alcoholic  solution.  For 
use  dilute  this  with  one  half  the  quantity  of 
water, 


242  THE  TECHNOLOGY  OF 

12.  CEMENT  FOR  GLYCERINE  MOUNTS. 
Pure  Venetian  turpentine  is  to  be  poured  into 
some  melted  wax  upon  a  water  bath  until  a  por- 
tion taken  out  on  a  glass  rod  becomes  at  once  stiff 
and  does  not  stick  to  the  hand.      (Dr.  Csokor, 

Wien.) 

% 
13.  CULTURE  FLUIDS. 

(vid.  F.  31.  46.  1. ) 

(1)  Liebig's  extract  of  meat 1  part 

Water 100 

(2)  Gelatine 3      parts 

Phosphate  of  ammonia 25    " 

Water 100 

(3)  Meat  extract 1      part 

Sugar 3 

Water       100 

(4)  Pasteur's  Fluid. 

Distilled  water 100  parts 

Cane  sugar  (pure) 10     " 

Ammonium  tartrate 1     " 

Ash  of  yeast 1     " 

(5)  Cohris  Fluid. 

Distilled  water  ....  100  ccm.  or  200  parts 
Ammonium  tartrate  .     .  1  gramme      20      " 
Potassium  phosphate .     .  0.5     «*  20      " 

Magnesium  sulph.  cryst.    0.5     "  10      " 

Calcium  phosphate  (tri- 

basic)  0.5     "  0.1      " 

Fluids  (4)  (5)  are  not  suitable  for  the  cultiva- 
tion of  pathogenic  organisms.     In  making  broths, 


BACTERIA  INVESTIGATION.  24S 

select  as  fresh  meat  as  possible,  allow  half  an 
hour's  boiling  for  each  pound,  and  calculate  to  have 
sufficient  water  to  yield,  ultimately,  one  pint  of 
broth  to  each  pound.  When  boiled,  allow  to  cool, 
skim  off  fat,  and  neutralize  with  carbonate  of 
sodium,  filter  through  a  sterilized  filter  into  steri- 
lized flasks.  If  the  broth  is  not  clear  after  once 
filtering,  repeat ;  and,  if  upon  standing,  a  sediment 
appears,  decant,  or  boil  with  the  white  of  an  egg, 
and  filter. 

14.    EOSIN-ELEMATOXYLIN. 

(1)  Eosin 5 

Distilled  water 100 

Alum 2.5 

Glycerine 100.0 

(2)  Haematoxylin .5 

Absolute  alcohol .  100 

Mix  (1)  and  (2),  After  three  days,  during 
which  the  mixture  is  exposed  to  the  light,  add 
glacial  acetic  acid  2  per  cent.  (Niesser.} 

15.  EOSIN. 

This  dye  appears  in  commerce  as  eosinate  of 
potash.  In  a  saturated  solution  of  the  salt  a  pure 
eosinic  acid  will  be  precipitated,  if  an  acid  is 
added.  If  the  precipitate  is  collected  upon  a 
filter,  and  dissolved  in  strong  alcohol  it  gives 
a  solution  of  pure  eosinic  acid. 

The  ordinary  eosinate  of  potash  is  used  in  a  5 


244  THE   TECHNOLOGY  OF 

or  10  per  cent  aqueous  solution,  of  which  one 
adds  some  drops  to  the  fluid  containing  the  sec- 
tions. It  is  better  to  mount  sections  stained  in 
eosin  in  glycerine  containing  alum. 

16.  FUCHSIN. 

Used  by  Rindfleish  in  a  concentrated  alcoholic 
solution. 

Used  by  Lichtheim  in  a  concentrated  aqueous 
solution. 

Used  by  Pfuhl-Petri  in  following  solution  :  — 

Saturated  alcoholic  solution  of  Fuchsin     10  ccm. 
Distilled  water 100  ccm. 

BurrilVs  Solution. 

Glycerine 20  parts 

Fuchsin       3      " 

Aniline  oil 2      " 

Carbolic  acid 2      " 

A  slight  variation  in  the  quantities  does  not  mat- 
ter, but  the  glycerine  should  not  be  decreased. 
Mix  and  shake  carefully.  Prepare  a  quantity  of 
the  solution  in  advance,  as  it  does  not  deteriorate 
as  do  alcoholic  solutions. 

Gradle's  Solution,  recommended  by  Hartzell. 

Carbolic  acid 15  minims 

Distilled  water \  fluid  oz. 

Dissolve,  and  add  alcoholic  solution  of  fuchsin,  \  fluid 
drachm. 


BACTERIA    INVESTIGATION.  245 

17.    FUCHSIN- ANILINE-OIL. 

Add  10  or  20  drops  of  a  concentrated  alcoholic 
solution  of  fuchsin  to  10  ccm.  of  aniline-water. 
Used  by  Ekrlich  for  tubercle  Bacilli  and  by  Baum- 
garten  for  lepra  Bacilli. 

18.   GENTIAN- VIOLET. 

(a)  Weigert's  solution. 

Distilled  water 90   grams 

Absolute  alcohol 10 

Caustic  ammonia 0.5 

Gentian  violet 2. 

Mix,  and  filter. 

(b)   Cose  and  Simon's  Solution. 
Gentian- violet  (or  fuchsin)  ....    2  grams 
Alcohol  (90  per  cent)       .     .         .    .    5      " 
Saturated  aniline-water  ....      100      " 

Jfddtzers  solution. 

Make  a  saturated  solution  with  90  per  cent 
alcohol. 

19.  ANILINE-GENTIAN-VIOLET. 

(a)  Balmer-FraenzeTs  solution 

Pure  aniline  oil,    1  ccm.  i 

£  =  Aniline  water 
Water      ...    24  ccm.  5 

Shake  together,  and  pass  through  a  wet  filter- 
paper.  To  the  clear  filtrate  add  one  half  gram 
of  finely  powdered  gentian-violet.  Dissolve  by 


246  THE  TECHNOLOGY  OF 

stirring  with  a  stick.       Filter  just  before  using. 
(2  grams  to  100  ccm.) 

(b)  JEJhrlich's  solution  of  Gentian-violet  or  Fuchsin. 
To  saturated  aniline  water  10  ccm.,  add  concen- 
trated alcoholic  solution  of  either  the  above  (10- 
20  drops)  until  opalescence  occurs  (showing  satu- 
ration). Gentian-violet  is  also  used  in  concen- 
trated alcoholic  solutions ;  also  in  concentrated 
aqueous  solutions  (Lichtheim).  Kaatzer  uses  10 
drops  of  a  saturated  alcoholic  (90  per  cent)  solu- 
tion of  gentian-violet  to  10  cc.  aniline  water. 
Shake  well,  and  filter.  Gentian- violet  gives  a 
good  double-staining  with  vesuvin. 

20.  KLEB'S  GLYCERINE- JELLY  FOR  MOUNTING. 

To  be  used  warm,  or  a  piece  laid  on  the  slide 
and  this  warmed.  All  air-bubbles  must  be  driven 
off.  Its  advantages  are  that  it  hardens  in  cooling, 
so  that  a  slide  may  be  cleaned  andr  finished  at  once. 

Take  best  clear  gelatine,  10  grams.  Allow 
this  to  swell  up  in  distilled  water ;  throw  away 
the  water  left,  and  dissolve  the  swollen  glue  by 
gentle  warming.  Then  add  10  grams  of  gly- 
cerine and  a  few  drops  of  phenol,  to  prevent  the 
formation  of  mould. 

21.  GLYCERINE. 

Use  pure  glycerine,  entirely  free  from  acid 
reaction.  Glycerine  is  not  adapted  for  mounting 


BACTERIA  INVESTIGATION.  247 

Bacteria  which  have  been  stained  in  any  of  the 
aniline  dyes,  with  the  exception  of  Bismarck- 
brown.  Microbes  stained  in  Bismarck-brown  and 
mounted  in  glycerine  make  the  best  preparations 
for  photographing. 

22.  BOEHMER'S  HJEMATOXYLIN. 

(1)  Dissolve  0.35   parts  of  crystallized  haema- 
toxylin  in  ten  parts  of  absolute  alcohol. 

(2)  Dissolve  1    part  of  alum  in   30  parts  of 
distilled  water.     Add  the  first  solution  gradually 
to   the   second,   until    a    beautiful   violet    is   de- 
veloped. 

Cose  and  Simon  recommend  the  following  solu- 

o 

tion.  Make  a  2  per  cent  hydro-alcoholic  solution 
of  haematoxylin  and  a  3  per  cent  solution  of  am- 
monia alum.  A  few  drops  of  the  hsematoxylin 
added  to  a  small  quantity  of  the  ammonia  alum 
solution  gives  in  the  course  of  a  few  minutes  a 
beautiful  violet  color. 

23.  METHYL-BLUE. 

(a)  Koch's  Solution. 

Take  distilled  water  200  cctm. 

Concentrated  alcoholic  solution  of  Methyl-blu^ 
10.  Shake  well,  and  add  — 

Ten  per  cent  solution  of  caustic  potash  0.2. 
Allow  the  mixture  to  stand  for  a  day.  It  should 
give  no  sediment.  Filter. 


248  THE  TECHNOLOGY   OF 

(b)  FrdnkeTs  Solution,  For  double-staining. 

Alcohol 50.0 

Water 30.0 

Nitric  acid 20.0 

Saturate  with  methyl-blue,  and  filter.  This 
solution  removes  from  the  matrix  in  which  the 
Bacteria  lie  the  dye  first  used,  and  replaces  it  with 
blue. 

(c)  Schutz's  Solution. 

Caustic  potash  solution  (1-10,000,  j 

Absolute  alcohol,  >  Equal  parts 

Solution  of  methyl-blue,  ) 

(d)  Watson  Cheyne's  Solution. 

Distilled  water 100  ccm. 

Saturated  alcoholic  solution  of  methyl- 
blue    20  ccm. 

Formic  acid 10  minims 

(Practitioner,  April,  1883,  p.  258.) 

24.  METHYL-GREEN  AND  MALACHIT-GEEEN. 

(a)  Of  the  former,  use  saturated  aqueous  solu- 
tion (Subbotiri). 

(b)  Of  the   latter,    use   a   saturated    alcoholic 
solution  (Pfuhl  and  Petri) . 

25.  METHYL-VIOLET. 

Used  by  Baumgarten  and  Fraenkel  in  a  saturated 
alcoholic  solution. 


BACTERIA    INVESTIGATION.  249 

Koch's  Formula. 

Take  of  pure  aniline  5  cc.,  shake  repeatedly 
with  distilled  water.  In  half  an  hour  3  or  4  per 
cent  of  the  aniline  is  dissolved,  the  rest  settles  to 
the  bottom.  This  mixture  is  filtered,  and,  if  not 
absolutely  clear,  must  be  filtered  again.  Prepare 
new  each  time. 

To  100-150  cc.  of  absolute  alcohol  add  20 
grams  of  dry  methyl- violet,  allow  to  stand  sev- 
eral days,  shake  repeatedly.  Fuchsin  may  be 
used  instead. 

11  cc.  of  the  alcoholic  solution  of  methyl-violet 
is  mixed  with  100  cc.  of  the  aniline-water,  and  the 
staining  fluid  is  ready  for  use.  Add  a  crystal  of 
thymol  to  keep  it,  and  filter  before  use. 

26.  MAGENTA. 

Magenta  crystals 2  grams 

Pure  aniline  oil 3  ccm. 

Alcohol  (sp.  gr.  0.830) 20     " 

Water 20     " 

Pulverize  the  crystals,  dissolve  in  the  aniline- 
oil  and  alcohol,  and  lastly  add  the  water  (Gibbs). 

27.  NIGROSIN  OR  ANILINE-BLACK. 

Use  a  strong  aqueous  solution ;  wash  sections 
in  alcohol,  and  mount  either  in  glycerine  or  Can- 
ada balsam  (JErrera). 


250  THE  TECHNOLOGY  OF 

28.   ORSEILLE. 
Orseille  extract  is  derived  from  Rocella  tinctoria. 

Absolute  Alcohol .    20  cctm. 

Glacial  acetic  acid  (sp.  gr.  1.070)    .     .       5     " 
Distilled  water 40     " 

Add  as  much  orseille  extract  as  will  form  a  satu- 
rated dark  red  fluid,  which  must  be  filtered  two  or 
three  times,  until  a  clear  ruby  fluid  is  obtained. 
This  makes  a  good  stain  for  the  tissue  containing 
Bacteria,  as  it  stains  cell-substance  but  not  nuclei. 
Mount  in  levulose  (vid.  Virchow's  Archiv,  Bd. 
71.)  (Wedl.) 

29.  OIL  OF  CLOVES. 

While  this  is  most  commonly  used  for  clearing 
stained  tissues,  it  may  be  replaced  by  other  ethe- 
real oils,  e.  g.,  cedar,  origanum,  cinnamon,  berga- 
mot,  lavender,  anise,  or  by  xylol  or  creosote. 

30.   OSMIC  ACID. 

This  is  a  volatile,  strong-smelling  crystalline 
substance,  very  irritating  to  mucous  membranes. 
The  breathing  of  the  fumes  must  be  positively 
avoided.  In  commerce  it  comes  in  small,  sealed 
glass  tubes,  each  holding  a  gram  or  a  half 
gram  of  the  greenish,  crystallized  acid.  It  is  used 
as  a  fixing  and  hardening  medium  in  aqueous 
solutions  of  1  to  500,  or  1  to  100.  The  tube 


BACTERIA    INVESTIGATION.  251 

should  be  broken  inside  a  bottle  containing  the 

o 

proper  quantity  of  distilled  water,  by  means  of 
a  glass  rod.  The  solution  must  be  kept  from  the 
light,  or  it  will  be  rapidly  reduced. 

31.  PEPTONE  SOLUTION. 

Peptone  (chemically  pure) 5.9 

Bi-sodic  phosphate 10. 

Lactate  of  ammonia 5. 

Liebig's  beef  extract 5. 

Sugar 20. 

Distilled  water 1000. 

Peptone,  being  a  changeable  product,  is,  if  not 
fresh,  best  prepared  especially  for  the  purpose, 
e.  g.,  allow  500  grams  of  lean  beef,  2  litres  of 
water,  and  1  gram  of  salt  to  simmer  for  four 
hours,  in  an  uncovered  vessel.  Then  close  it, 
and  heat  for  one  hour  to  100°  C."  Cool,  and  skim 
off  the  fat;  neutralize  carefully,  and  filter. 

Buchner's  Fluid. 

Leibig's  extract 10  parts 

Peptone 8     " 

Water 1000      " 

32.     PlCROCARMINE. 

Add  aqua  ammonia,  4  grams,  to  carmine,  2 
grams,  and  allow  it  to  stand  for  twenty-four 
hours  in  a  damp  place,  and  then  add  200  grams 
of  picric  acid.  Allow  the  whole  to  remain  for 
twenty-four  hours  longer,  until  all  is  dissolved 


252  THE   TECHNOLOGY   OF 

that  will  dissolve.  Filter,  and  to  the  filtrate  add 
a  small  quantity  of  acetic  acid,  until  it  becomes 
turbid.  After  twenty-four  hours  more  there  is 
a  precipitate,  and  the  filtered  fluid  also  remains 
turbid.  Now  add  ammonia,  drop  by  drop,  and 
allow  the  solution  each  time  to  remain  for  twenty- 
four  hours,  until  at  length,  in  the  course  of  a  few 
days,  it  remains  entirely  clear.  If  the  neutral 
solution  stains  too  yellow,  add  a  little  acetic  acid  ; 
if  too  red,  a  little  ammonia.  (  Weigert.) 

33.     PlCROCARMINATE    OF    AMMONIA. 

Pour  into  a  saturated  solution  of  picric  acid  a 
strong  ammoniacal  carmine  solution  until  it  be- 
comes turbid.  Evaporate  this  mixture  to  one 
fifth  of  its  original  volume  ;  filter.  Now  evaporate 
the  filtrate  to  dryness,  and  add  the  red  powder 
resulting  each  time,  before  use,  to  about  one  hun- 
dred parts  of  water,  or  allow  the  original  solution 
to  dry  in  the  open  air,  in  an  evaporating  dish  ; 
dissolve  the  residue  in  a  little  water,  filter,  and 
add  to  the  filtrate  some  carbolic  acid,  to  prevent 
fermentation .  ( Ranvier. ) 

34.  ACETATE  OF  POTASH  MOUNTING  FLUID. 

This  is  simply  a  strongly  concentrated  solution 
of  acetate  of  potash.  It  behaves  much  as  glycer- 
ine, does  not  dry  at  the  edges,  and  is  less  refractive. 
(Refractive  index  for  the  line  D,  1 ,  370.  Oh.  Soret.} 


BACTEEIA    INVESTIGATION.  253 

Many  aniline-stained  preparations  of  Bacteria  keep 
well  in  this  solution.  (Max  Schultze.) 

35.  CAUSTIC  POTASH  SOLUTIONS. 

(a.)    Caustic  potash  fus 1  gram 

Absolute  alcohol 100  ccm 

Allow  to  stand  twenty-four  hours,  until  the  alco- 
hol is  saturated.  Decant,  and  mix  with  ten  times 
the  volume  of  pure  alcohol. 

(&.)     Caustic  potash  fus 20  parts 

Water 100 

(c.)  Baumgarten  uses  carbonate  of  potash  in 
his  method,  in  a  half  saturated  solution  for  decol- 
orizing. 

(c?.)    lodo-Iodide  of  Potash,  solution  for  decolor- 
izing. 

Iodine 1.0 

Iodide  of  potash 2.0 

Water 300.0 

3  6 .    ROS  ANILINE-CHLORHYDRATE . 

Rub  up  2  grams  of  rosanilin-chlorhydrate  with 
1  gram  of  methyl-blue,  and  dissolve  slowly  in  15 
ccm.  of  alcohol.  To  this  add  3  ccm.  of  aniline 
oil  and  15  ccm.  of  distilled  water.  Keep  in  a 
stoppered  bottle.  (Gibbes.) 

37.   ACID  FUCHSIN  (Rosanilinsaures  Natron). 
A  saturated  aqueous  solution  is  used. 


254  THE   TECHNOLOGY   OF 

« 

38.  VESUVIN. 

This  combines  well  for  double  staining  with 
methyl-blue  or  gentian-violet. 

FraenkeTs  Solution,  for  double  staining. 

Alcohol 70.0 

Nitric  acid 30.0 

Saturate  with  vesuvin,  and  filter. 

39.  PLASTER  OF  PARIS  MIXTURE  FOR  FILTER. 

Water 46. 

Plaster-of-Paris  (such  as  is  used  for  modelling)  52.4 
Asbestos 1.6 

Mix  carefully,  adding  the  plaster  gradually. 

40.  CLEANING  FLUIDS  FOR  SLIDES  AND  COVERS. 

(Hanaman's  Formula.}  — To  a  cold  saturated  so- 
lution of  bichromate  of  potash,  add  |  of  its  bulk 
of  strong  sulphuric  acid,  (care  must  be  taken  on 
account  of  the  heat  and  vapors  evolved).  Journ. 
Koy.  Mic.  Soc.  i.  (1878)  p.  295.  Amer.  Natu- 
ralist, XII.  p.  573. 

(Gibbs's  Method}. — Place  the  cover-glasses  in 
strong  sulphuric  acid  for  an  hour  or  two.  Wash 
well,  until  the  drainings  give  no  acid  reaction ; 
wash  first  with  methylated  spirits,  and  then  with 
absolute  alcohol,  and  wipe  carefully  with  an  old 
silk  handkerchief.  Journ.  Koy.  Mic.  Soc.  iii. 
(1880)  p.  392. 


BACTERIA    INVESTIGATION.  255 

(Seller's  Method.)  —  New  slides  and  covers  are 
placed  for  a  few  hours  in  the  following  solution  :  — 

Bichromate  of  potash 2  ounces 

Sulphuric  acid 3  fluid       " 

Water 25    " 

Wash  with  water,  the  slides  may  be  simply 
drained  dry ;  the  covers  may  be  wiped  dry  with  a 
linen  rag.  Slides  and  covers  that  have  been  used 
for  mounting  either  with  balsam  or  a  watery  me- 
dium are  treated  as  follows  : — 

The  covers  are  pushed  into  a  mixture  of  equal 
parts  of  alcohol  and  hydrochloric  acid,  and,  after  a 
few  days,  are  put  into  the  bicromate  solution  and 
treated  like  new  ones.  The  slides  are  scraped  free 
of  the  mounting  medium  and  put  directly  into  the 
bichromate  solution.  Ibid.  p.  508. 

41.  SOAP  IMBEDDING  MASS. 
Take  good  white  soap,  cut  it  up  into  thin  slices, 
and  put  them  to  dry  in  the  sun  for  some  days, 
until  they  become  white.  The  slices  are  then  to 
be  rubbed  up  to  a  fine  powder,  which  is  mixed 
with  spirit  to  the  consistency  of  porridge.  Now 
mix  the  porridge  with  alcohol  and  glycerine  in 
such  proportions  that  the  whole  shall  contain,  for 
every  10  parts  by  weight  of  the  soap,  22  parts  of 
glycerine,  and  35  parts  of  alcohol,  (90  per  cent). 
Let  the  whole  simmer  until  there  is  obtained  a 
perfectly  transparent,  syrupy,  somewhat  yellow 


256  THE  TECHNOLOGY  OF 

fluid.  The  objects,  previously  dehydrated  in  al- 
cohol, are  imbedded  in  this  mass  in  the  usual 
manner.  The  mass  may  be  removed  from  the 
sections  either  by  means  of  water,  or  of  very  dilute 
alcohol.  It  has  the  following  advantages.  1.  It 
is  transparent.  2.  It  adapts  itself  perfectly  to  the 
objects.  3.  It  cuts  remarkably  well.  (Salensky.} 

42.  CELLOIDIN  IMBEDDING  MASS. 
Celloidin  is  a  preparation  of  pure  pyroxylin. 
It  is  manufactured  by  the  Chemische  Fabrik  auf 
Actien  (vorm.  E.  Sobering),  Berlin,  N.  Fenstrasse, 
11.  12.  It  may  be  obtained  by  mail  by  writing 
to  Schering's  Griine  Apotheke,  Wittick  and  Ben- 
kendorf,  Berlin,  N.  Chausse'e-Strasse,  No.  19  [or 
from  wholesale  dealers  in  the  United  States] .  It 
is  stated  to  be  prepared  with  the  purest  pyroxylin, 
and  to  be  always  of  uniform  composition.  It  is 
sent  in  the  form  of  tablets  of  a  tough,  gelatinous 
consistency  and  slightly  milky-white  transparency. 
These  tablets  have  exactly  the  consistency  that  is 
required  for  section-cutting.  They  contain  20  per 
cent  of  pure  pyroxylin.  Celloidin  is  soluble  in 
all  proportions  in  ether  and  alcohol.  It  is  free 
from  acids.  Merkel  and  Schifferdecker  speak  of 
celloidin  as  an  imbedding  medium,  as  "  performing 
more,  less  hurtful  to  the  tissues,  and  easier  toman- 
age,  than  any  other  known  imbedding  mass."  Cut 
the  celloidin  into  small  pieces,  and  dissolve  in 


BACTEEIA    INVESTIGATION.  257 

equal  parts  of  absolute  alcohol  and  ether.  The 
tissues  to  be  imbedded  are  thoroughly  soaked  in 
absolute  alcohol,  from  which  they  are  brought  into 
the  celloidin  (in  a  well-closed  vessel),  and  remain 
there  until  thoroughly  impregnated  (from  a  few 
minutes  to  eight  days  or  more,  according  to  thick- 
ness). If  the  objects  contain  cavities  that  it  is 
desired  to  fill,  it  is  best  to  use  a  thinner  and  there- 
fore more  penetrating  solution  of  celloidin.  When 
soaked,  remove  the  preparations  to  a  paper  tray 
(or  simply  a  small  piece  of  leather),  surround 
them  with  celloidin ;  wait  a  few  minutes,  until  a 
skin  has  formed  on  the  celloidin,  and  throw  them 
into  alcohol  of  82°Richter  (a  considerable  quantity 
of  alcohol  should  be  taken).  After  twenty-four 
hours  the  preparations  on  leather  are  generally  fit 
to  be  cut,  whilst  those  in  paper  trays  may  have 
the  paper  removed  and  be  put  back  in  the  alcohol 
for  twenty-four  hours  more.  The  preparations 
may  remain  in  the  alcohol  for  any  length  of 
time  without  harm.  Sections  are  cut  with  a  knife 
moistened  with  common  alcohol ;  they  are. floated 
into  either  water  or  alcohol.  Without  any  further 
manipulation  they  may  now  be  stained  with  the 
usual  staining  agents,  just  as  if  they  were  not  im- 
bedded. Dehydrate  in  95  per  cent  alcohol  (not 
absolute,  or  the  celloidin  will  be  dissolved)  clear 
with  oil  of  bergamot,  sandal  wood,  or  origanum, 
(the  last  by  preference) ,  and  mount  in  balsam  or 


258  THE  TECHNOLOGY   OF 

castor  oil.  Oil  of  cloves  should  not  be  employed, 
because  it  dissolves  the  celloidin. 

Sections  may  be  cleared  and  mounted  in  glycer- 
ine, which  suffices  to  make  the  celloidin  as  clear  as 
glass. 

Instead  of  hardening  in  alcohol,  the  tissues,  after 
being  thoroughly  impregnated  with  celloidin,  may 
be  allowed  to  dry  under  a  bell-glass,  or  may  be 
brought  into  chloroform.  Under  the  influence  of 
this  reagent,  the  celloidin  coagulates  into  a  mass 
having  the  consistence  of  wax,  but  having  also  an 
elasticity  that  renders  it  unbreakable,  and  having 
besides  the  precious  quality  of  being  admirably 
transparent,  and  possessing  exactly  the  index  of 
refraction  of  glass.  (From  Microtomist's  Vade 
Mecum.  See  p.  197-199.) 

43.  BRUN'S  MOUNTING  MEDIUM. 

Glycerine 10  parts 

Glucose  of  commerce 40     " 

Spts.  of  camphor 10     *' 

Water 140     " 

Mix  and  filter.  The  advantage  of  this  medium 
is  that  it  possesses  the  refractive  index  1.37  in  the 
yellow  ray,  an  index  corresponding  to  that  of  the 
substances  composing  sputum  (albumen  1.36, 
saliva  and  mucus  1.34,  pus  1.39,  Robin).  Ordi- 
nary Canada  balsam  has  a  very  high  index,  1.53  ;  in 
which  Bacteria  are  poorly  defined ;  colorless  oils, 


BACTERIA    INVESTIGATION.  259 

for  example,  that  of  rape  or  castor  are  preferable, 
but  their  index  is  also  too  high,  1.48. 

44.  GAUTIER'S  KED-LEAD  CEMENT. 

Crystalized  boracic  acid 8  parts 

Silicic  acid 2      " 

Red  lead 12      " 

45.  MIQUEL'S  NUTRITIVE  PAPER. 

Pour  upon  a  large  sheet  of  black  or  gray  paper, 
boiling  lichen  jelly  (F.  46a),  and  spread  it  out 
uniformly,  so  that  in  the  wet  state  it  has  a  thickness 
of  2-3  mm.  This  done,  dry  it  rapidly  in  an  oven, 
at  40°.  In  a  few  hours  the  sheet  of  paper  is  dry. 
It  is  thin,  flexible,  it  never  presents  cracks,  and 
resembles  altogether  the  photographic  paper  cov- 
ered with  bromo-gelatine  emulsion,  invented  by 
Dr.  Maddox.  This  nutritive  lichen  jelly  paper 
may  be  preserved  indefinitely  with  all  its  qualities, 
it  being  only  necessary  to  place  it  in  a  dry  drawer. 
At  the  moment  of  making  the  experiment,  the 
paper  is  sterilized  by  being  suspended  in  an 
atmosphere  of  vapor  at  110°  C.  This  is  accom- 
plished without  its  running,  and  it  is  scarcely 
any  swollen.  Dr.  Maddox  proposes  that  col- 
lodion films  be  used  instead  of  paper,  on  ac- 
count of  the  greater  transparency  and  absence 
of  structure. 


260  THE  TECHNOLOGY  OF 

46.  SOLID  CULTURE  MEDIA. 
(a)  MiqueTs  Lichen  Jelly. 
Digest  in  one  litre  of  beef  broth,  25  to  30  grams 
of  Irish  moss  (Chondrus  crispus),  and  pass  the 
resulting  decoction  through  a  sieve  which  retains 
the  swollen  leaves.  After  neutralization  and  a 
short  boiling,  filter  the  broth  through  bolting-cloth. 
Upon  cooling  it  forms  a  strong  jelly.  To  obviate 
the  loss  of  broth  which  remains  in  the  swollen 
leaves,  Miquel  makes  in  his  laboratory  a  lichen 
jelly  with  water,  this  he  dries  and  adds  to  broth 
in  proper  proportions,  about  1  per  cent.  This 
nutritive  jelly  possesses  the  following  advantages. 

1.  It  melts  only  between  55°  and   60°  C.,  which 
permits  of  the  cultivation  of  such  organisms  as  re- 
quire for  their  development  elevated  temperatures. 
Ordinary  nutritive   gelatines   melt  before  30°  C. 

2.  It    remains   without   alteration    or   losing   its 
power  of  solidifying  when  exposed  to  a  tempera- 
ture of  110°  C.,  for  rigorous  sterilization.     Gela- 
tine, on  the  contrary,  is  reduced  under  such  con- 
ditions to  a  turbid  broth,  which  remains  fluid  on 
cooling.     Dr.  Miquel  does  not  favor  those  micro- 
graphic  methods,  introduced  from   Germany,   by 
which  one  is  recommended  to  use  an  intermittent 
heat  of  100°  C.     All  germs  are  not  destroyed  by 
this  degree  of  moist  heat,  and  if  one  obtains  gela- 
tines having  the  appearance  of  being  sterilized,  it 


BACTERIA    INVESTIGATION.  261 

is  for  the  simple  reason  that  the  germs,  being 
buried  in  the  gelatine  and  deprived  of  oxygen, 
are  under  bad  conditions  for  giving  a  visible 
growth. 

(b)  Hydrocele  Fluid.   (Koch.) 

Use,  for  tapping,  a  thoroughly  sterilized  trocar 
and  canula,  connected  by  a  sterilized  rubber  tube 
with  a  flask  also  sterilized.  The  collected  fluid  is 
then  in  its  turn  sterilized,  by  exposure  to  a  tem- 
perature of  58°-62°  C.,  for  three  to  five  hours,  for 
five  or  six  consecutive  days.  This  may  be  con- 
sidered accomplished  if  the  fluid  remains  limpid  in 
the  incubator  at  32°-38°  C.,  for  several  weeks. 
This  limpid  fluid  may  be  used  in  fluid  cultures 
alone,  and  in  solid  cultures  mixed  with  gelatine,  or 
it  may  be  made  solid  in  the  same  manner  as  blood 
serum  by  heating  it  gradually  up  to  60°-70°  C. 
In  the  course  of  an  hour  it  solidifies.  It  is  impor- 
tant to  proceed  gradually  in  this  process,  as  there 
is  danger  of  its  losing  its  limpidity,  and  the  heat- 
ing should  be  done  in  vessels  in  which  it  is 
to  be  used,  as  it  cannot  be  again  liquefied.  A 
better  method  of  rendering  hydrocele  fluid  and 
blood  serum  solid  through  evaporation  is  to 
expose  them,  in  properly  protected  sterilized 
test  tubes,  to  a  heat  of  32°-38°  C.  for  several 
weeks.  If  prepared  in  this  way,  the  limpidity  is 
not  lost. 


262  THE  TECHNOLOGY   OF 

(c)  Blood  Serum.  (Koch.) 
Blood  of  a  healthy  sheep  is  drawn  from  the 
carotid  artery,  by  means  of  a  canula,  into  a  flask, 
with  which  it  is  connected  by  a  rubber  tube,  the 
whole  apparatus  being  first  thoroughly  sterilized. 
After  standing  twenty-four  hours,  or  until  a  firm 
clot  has  formed,  the  serum  is  drawn  off  by  means 
of  a  sterilized  glass  siphon,  one  end  of  which  passes 
through  the  cotton  plug  in  the  flask  containing  the 
blood,  and  the  other  through  the  plug  in  the  ves- 
sel intended  for  the  reception  of  the  serum.  The 
serum  is  then  sterilized,  and,  if  desired,  rendered 
solid  by  the  same  process  as  that  used  for  hydro- 
cele  fluid. 


Agar  Agar.1 
This  substance  occurs  in  commerce  in  bunches 
of  thin,  shrivelled,  transparent  strips.  It  is  pre- 
pared for  use  by  soaking  over  night  in  salt  water 
(1-6),  and  then  dissolved  by  the  aid  of  heat. 
After  being  filtered  and  neutralized,  it  is  mixed 
with  some  nutritive  material.  Klein  says  that 
among  all  solid  media  he  finds  a  mixture  of  agar 
agar  and  peptone  the  best.  He  prepares  it  by 
mixing  peptone  with  the  filtered  agar  agar  solu- 
tion, boils  it  repeatedly  for  thirty  minutes  at  a 

1  To  be  had  of  Dr.  Georg  Griibler,  17  Durfour  Strasse,  Leipzig; 
Physiologische-chemische  Laboratorium,  or  of  Messrs.  Christy  & 
Co.,  155  Fenchurch  Street,  London. 


BACTEEIA    INVESTIGATION.  263 

time,  and  finally  obtains  a  sterile  transparent 
mass,  which  remains  solid  up  to  45°-  50°  C.  It 
becomes  liquid  at  higher  temperatures,  and,  in 
case  of  necessity,  may  be  again  subjected  to  boil- 
ing. Before  considering  it  as  perfectly  sterile,  it 
ought  to  be  kept,  like  all  other  materials,  for  from 
several  days  to  several  weeks  in  the  incubator,  at 
32°-  38°  C.  If  quite  limpid  after  this  time  it 
may  be  considered  as  sterile. 

(e)    Gelatine. 

The  best  gelatine  is  cut  up  into  strips  and  soaked 
in  distilled  water  over  night  ( 1  part  of  gelatine  to 
6  of  water) ,  and  is  then  melted,  thoroughly  neu- 
tralized, and  filtered  through  sterilized  bolting 
cloth.  If  it  is  not  clear,  it  is  boiled  with  some 
white  of  egg  and  again  filtered.  The  fluid  gela- 
tine is  then  mixed  with  half  its  bulk  of  broth, 
peptone  solution,  or  beef-extract  solution,  so  that 
there  is  1  part  of  gelatine  in  9  parts  of  fluid,  or 
11^  per  cent  of  gelatine.  This  mixture  is  boiled 
repeatedly,  and  treated  like  fluid  culture  media, 
as  described  above.  It  may  be  kept  on  hand  as  a 
stock,  either  with  the  addition  of  the  peptone,  etc., 
or  as  simple  gelatine,  which  can  be  added  to  any 
particular  nourishing  material  when  desired. 


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